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* Institut für Klinische Chemie und Laboratoriumsmedizin, 48149 Münster, Germany; and
Institut für Immunologie, Universität Witten/Herdecke, Witten 58453, Germany
1Correspondence: Institut für Klinische Chemie und Laboratoriumsmedizin, Albert-Schweitzer-Str. 33, 48149 Münster, Germany. E-mail: brandt{at}uni-muenster.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: F-actin • gelsolin • epidermal growth factor • HUVEC
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4
5
6
7
8)
. The clinical effect of
c-erbB-2 was further substantiated by the fact that c-erbB-2 expression
in human breast cancer cells appeared to be capable of enhancing
metastatic potential in vivo and invasiveness in
vitro (9
10
11)
.
Although a ligand of c-erbB-2 in human cancers is still unknown, the
known ligands (e.g., EGF) of the other erbB receptors induce tyrosine
phosphorylation with c-erbB-2 by heterodimerization (12)
.
Results from molecular conformational energy calculations of c-erbB-2
and EGFR tyrosine kinase domains indicate that heterodimers would be
preferred over homodimers (13
, 14)
. Furthermore,
down-regulation experiments of c-erbB-2 and EGFR by the intracellular
expression of specific antibodies have shown that heterodimer formation
follows a strict hierarchy and c-erbB-2 is the preferred counterpart of
the other erbB family members in heterodimerization (12)
.
It was also observed that signals elicited by growth and
differentiation factors (EGF, NDF, BTC, HB-EGF) were amplified or
prolonged by c-erbB-2 phosphorylation (15
16
17)
. Therefore,
an important role of c-erbB-2 in the lateral transmission of signals
between the erbB receptors was suggested (16)
. Thus, in a
nude mouse model, cells expressing both c-erbB-2 and EGFR most
frequently enhanced tumor formation and developed higher tumor masses
than did cells expressing any other combination of the erbB receptors
(18)
.
As a consequence of the frequent detection of c-erbB-2-positive
staining cell clusters in the peripheral blood of breast cancer
patients (19)
, we established an in vitro model
for the capillary wall by growing human umbilical vein endothelial
cells (HUVECs) on porous membranes coated with basement membrane
extracellular matrix and thereby demonstrated that c-erbB-2-expressing
cell subpopulations within an individual primary breast cancer are of
high transendothelial invasiveness (20)
.
The present study aimed to substantiate the possible involvement of
c-erbB-2 in this transendothelial invasiveness and to investigate how
it is linked to the mode of cytoskeleton activity. Therefore, two
breast cancer cell lines (SKBR3 and MDA-MB-468) expressing different
levels of the c-erbB-2 receptor, and their counterparts genetically
modified for c-erbB-2 expression, were applied to the model system. In
the cell lines with transendothelial invasiveness, c-erbB-2/EGFR
heterodimerization was found constitutively. Furthermore, c-erbB-2 and
EGFR were colocalized at areas undergoing extreme shape change. The
state of actin assembly is affected by different actin-modifying
proteins, among which gelsolin has been demonstrated to modulate the
rate of migration of an individual cell (21
, 22)
. We found
that gelsolin was also mobilized via c-erbB-2/EGFR transphosphorylation
and released to a great extent around points at which the cell moves
through the pore. F-Actin was localized exclusively to gelsolin and
phosphorylated c-erbB-2 in transendothelial invasive cells. Therefore,
we believe that our study strongly suggests for the first time that
c-erbB-2, especially in a heterodimer with EGFR, is closely involved in
the signaling pathway that induces the alterations in cell morphology
required for metastatic cells to leave the capillary bed.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The
cells were grown on gelatin-coated flasks and passaged four to six
times in HUVEC culture medium. Breast cancer cell lines SKBR3 and
MDA-MB-468 were obtained from the American Type Culture Collection
(ATCC, Rockville, Md.) and cultured in Dulbecco's modified Eagle's
medium (DMEM) (ICN, Eschwege, Germany) supplemented with 2 mM
L-glutamine, antibiotic drugs, and 10% fetal calf serum. For culture
of transfectants, cells were grown under the same conditions except for
addition of 400 µg/ml Geneticin (G418) (Sigma, Deisenhofen, Germany)
to the culture medium.
Endothelial-extracellular matrix functional assay
Cell culture inserts with 8 µM porous polyethylene
terephthalate (PET) membranes were placed in 12-well plastic tissue
culture plates (Becton Dickinson Labware, Franklin Lakes, N.Y.). The
membranes were coated with basement membrane extracellular matrix (ECM)
(Harbor Bio-Products, Norwood, Mass.) at a concentration of 125
µg/cm2 by drying an appropriate ECM dilution
overnight under a laminar flow hood. Dried ECM was rehydrated with 500
µl HUVEC culture medium, which contained equal volumes of Iscove's
modified Dulbecco's medium (IMDM) and Ham's F12 nutrient mixture
(Life Technologies, Eggenstein, Germany) supplemented with 10 µg/ml
sodium heparin (Boehringer Ingelheim, Heidelberg, Germany), 5 µg/ml
transferrin, 2.5 ng/ml basic fibroblast growth factor (bFGF) (Sigma), 5
µM ß-mercaptoethanol, 2 mM L-glutamine (Life Technologies), 100
U/ml penicillin, 100 µg/ml streptomycin, 250 ng/ml amphotericin B
(Sigma), and 15% FCS (PAA Laboratories, Linz, Austria) for 1 h.
HUVEC were seeded onto the coated membranes in a concentration of
2 x 105 cells/well. After culturing for 2
days at 37°C in a humidified atmosphere of 5%
CO2, HUVEC formed confluent monolayers, which
were verified by panoptic staining (Diff-Quik, Baxter Healthcare Co.,
Miami, Fla.). Cell culture inserts were used for up to 3 days after
endothelial cells reached confluence. For the invasion assays of breast
cancer cell lines, cells were harvested with 0.25% trypsin-2 mM EDTA
(Life Technologies), adjusted to a density of 2 x
105 cells/ml with serum-free invasion medium
(DMEM with 2 mM L-glutamine, antibiotic drugs and 0.1% bovine serum
albumin; Sigma), and 2 x 105 cells were
placed onto the HUVEC monolayer on the ECM-coated membrane. The
invasion medium was placed into the wells under the bottom sides of the
membranes as well. Invasion assays were incubated for 48 h at
37°C in 5% CO2. At the end of the invasion
assay, the HUVEC monolayer and noninvading cells on the upper surface
of the membrane were removed by Q-tips and the membranes were
thoroughly rinsed with phosphate-buffered saline (PBS, pH 7.4).
Invading cells on the uncovered lower side of the membrane were fixed
in 4% paraformaldehyde and characterized using double or triple
immunocytochemistry (see below).
Microinjection of c-erbB-2 invasive cell lines
Cells were seeded onto coverslips 48 h prior to
microinjection. After starving the cells for 24 h (DMEM, 0.1%
FCS), they were microinjected under a Fluovert microscope (Leitz,
Bensheim, Germany) using the Microinjector 5242 and the
Micromanipulator 5171 (Eppendorf, Hamburg, Germany). The antibody
specific against phosphorylated c-erbB-2 (PN2A, kindly provided by Mike
Di Giovanna, Yale, New Haven, Conn.) was used at a concentration of 1
mg/ml in PBS.
Immunofluorescence studies
For immunofluorescence studies, the cells were fixed,
permeabilized, blocked with 10% human AB-serum (AB-serum for
serological reactions (Biotest, Dreieich, Germany) to inhibit
nonspecific staining, and incubated with up to two of the following
antibodies: mouse monoclonal anti-c-erbB-2- antibody (PN2A, kindly
provided by M.P. DiGiovanna, Yale, New Haven), rabbit polyclonal
anti-c-erbB-2 antibody (21N, Dako, Hamburg, Germany), anti-EGFR
antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.), anti-gelsolin
antibody (Biozol, München). The mouse antibody and the rabbit
antibodies were visualized using polyclonal LRSC-conjugated goat
anti-mouse immunoglobulin G (IgG) F(ab') fragments and Cy5-conjugated
sheep anti-rabbit antiserum, respectively, which were applied for 30
min at room temperature. Biotinylated anti-cytokeratin antibodies were
visualized using a streptavidin-Cy5 conjugate in 10% AB-serum before
antibody incubation of the cells. Actin was stained with
Phalloidin-FITC as the last step in the immunochemical staining
procedure. After rinsing with PBS for 5 min, the membranes were mounted
with aquamount on a thickness zero coverslip. Confocal laser scanning
was performed on a Leica True Confocal microscope (TCS) (Leica,
Bensheim, Germany). Immunofluorescence images were obtained using an
Argon laser at 488 nm or the combined Argon-Krypton laser at
wavelengths of 488, 568, and 635 nm. Emitted and reflected light was
passed through a beam splitter and four channels were simultaneously
detected (FITC fluorescence, LRSC fluorescence, Cy5 fluorescence, and
transmission).
Western blot analysis
For preparation of cell lysates, confluent cell monolayers were
washed three times with PBS and scraped from 75
cm2 tissue culture flasks in ice-cold lysis
buffer containing 150 mM NaCl, 5 mM EDTA, 10 mM Tris/HCl (pH 7.2),
0.1% sodium dodecyl sulfate (SDS), 0.1% sodium deoxycholate, 1%
Triton-X-100, and protease inhibitors (mixture tablets Complete;
Boehringer, Mannheim, Germany). After 15 min on ice, the lysate was
centrifuged at 4°C and 12,000 x g for 1 h.
Protein concentration was determined using the BCA protein assay
reagent (Pierce, Rockford, Ill.). Immunoprecipitation was performed
with Dynabeads (M280) coated with anti-mouse or anti-rabbit sheep
antibodies. Defined amounts of protein were electrophoresed on a 7.5%
SDS-polyacrylamide gel. After electrophoresis, proteins were
transferred onto a polyvinylidene difluoride (PVDF) membrane (Roth,
Karlsruhe, Germany) and unspecific binding sites were blocked with 10%
nonfat dry milk (De-Vau-Ge Gesundkostwerk, Lüneburg, Germany) in
PBS with 0.1% Tween 20. Blots were probed with mouse antibody c-neu
(Ab-3) for p185neu (Oncogene Research Products, Cambridge, Mass.),
rabbit Ab (1005) for EGFR (Sanza Cruz Biotechnology), and PY20 for
phosphotyrosine horseradish peroxidase-conjugated (Santa Cruz
Biotechnology), followed by incubation with horseradish
peroxidase-conjugated goat anti-mouse antibody and anti-rabbit
antibody, respectively (Amersham). The antibodies were used at a
concentration of 0.1 µg/ml. Bands were visualized by horseradish
peroxidase/hydrogen peroxide-catalyzed oxidation of luminol in the
enhanced chemiluminescence (ECL) system (Amersham).
Cell transfection
MDA-MB-468 and SKBR3 cells were stably transfected with the
plasmid vector pCVN/HER-2 (kindly provided by A. Ullrich, Martinsried,
Germany) and pcDNA3neo containing antisense ligated sequences of
c-erbB-2 using the lipofectin reagent (Life Technologies), as
described. For control experiments, cells were transfected with plasmid
vector pCVN and pcDNA3neo lacking the inserts. Two days after
transfection, cells were harvested and selected with 400 µg/ml G418.
For a second selection step, G418-resistant cells were positively
sorted using immunomagnetic beads (Dynabeads, Dynal, Hamburg, Germany)
coated with p185c-erbB-2-specific antibody c-neu (Ab-5) (Oncogene
Research Products). Sorted MHER2 cells were >99% p185c-erbB-2
positive as detected by FACS analysis (FACScalibur, Becton-Dickinson,
Heidelberg, Germany).
Phosphatidylinositol hydrolysis
Cells were washed three times with PBS and incubated for 3 h at 37°C in phosphate-free DMEM containing 20 µCi/ml of
[32P]orthophosphoric acid and 20 mmol/l HEPES.
Cells were stimulated with 100 ng/ml EGF; after 30 s, 60 s,
90 s, 120 s, and 5 min, the reaction was terminated by
removing the medium and scraping the cells off the dishes with chilled
0.7% NaCl by use of a rubber policeman. Extraction was performed with
chloroform/methanol/HCl (100:200:2). Cells were inhibited for PIP2
cleavage, performing the same protocol by adding U73122 (Sigma).
[32P]Phospholipids were developed in
chloroform/methanol/acetone/acetic acid/water (60:20:23:18:12, v/v)
with potassium oxalate-impregnated silica 60 plates (Merck, Germany).
Bands corresponding to PIP2 and PIP were cut out from the silica
plates, placed in scintillation fluid, and quantified by liquid
scintillation counting. The identities of labeled bands were determined
based on RF values obtained for authentic phospholipids (Sigma)
visualized by iodine staining, as described by Billiah and Lapetina
(23)
.
Flow cytometry
1–3 x 106 cells were incubated in a
total volume of 100 µl with 2 µl of anti-gelsolin rabbit polyclonal
antibody for 45 min on ice. Anti-c-erbB-2 oncoprotein (rabbit; Dako)
was used as a positive control. The rabbit antibodies were visualized
using polyclonal PE-conjugated goat anti-mouse IgG F(ab') fragments and
FITC-conjugated sheep anti-rabbit antiserum, applied for 30 min at room
temperature. Nonspecific staining was controlled by using mouse isotype
control antibodies of the same IgG subclasses and concentrations.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A) presented with a high transendothelial invasiveness (Fig. 1B
). In contrast, only a negligible number of MDA-MB-468
cells (MDAwt), which express high levels of EGFR and almost no
detectable c-erbB-2 (Fig. 1A
), penetrated the
endothelium-extracellular matrix layer (Fig. 1B
). To ensure
that differences in the migration and invasion capacity were due to
differences in the c-erbB-2 expression levels and did not depend on
other differentially regulated genes, genetically modified counterparts
of the cell lines were produced. We transfected low invasive MDAwt
cells with the plasmid vector pCVN/HER2 containing the full-length
wild-type human c-erbB-2 cDNA and SKBR3 cells with the plasmid
pcDNA3neo, which contained antisense ligated c-erbB-2 cDNA sequences,
to generate the c-erbB-2-positive MDA-MB-468/HER-2 (MHER2) cell line
and the SKBR3AS transfectant with low c-erbB-2-expression levels,
respectively (Fig. 1A
). The c-erbB-2-transfected cell line
MHER2 gained transendothelial invasiveness in a c-erbB-2-dependent
manner, whereas SKBR3AS cells were significantly less invasive after
antisense down-regulation of c-erbB-2 (Fig. 1A
). For
controls, MDA-MB-468/neo and SKBR3/neo cell lines were established by
transfection with the empty plasmid vectors, which did not interfere
with c-erbB-2 expression levels (Fig. 1A
). The mock
transfected cell lines MDAneo and SKBR3neo both displayed a
transendothelial invasiveness as even as their respective parental cell
lines, MDAwt and SKBR3 (Fig. 1B
).
|
F-Actin reorganization during transendothelial invasion is
accompanied by c-erbB-2 receptor redistribution
Cell migration comprises morphological polarization, membrane
extension, and traction forces, which are mandatory for the
transendothelial invasion process. To study c-erbB-2 receptors and
F-actin distribution in a moving cell, we applied the highly invasive
cell line SKBR3 to the in vitro system that mimics a blood
capillary structure (20)
. The size relationships of the
HUVEC monolayer (1)
, basement membrane layer
(2)
, and 8 µM PET membrane pores (3)
are
shown by transmission electron microscopy (Fig. 2
A).
|
In a nonmigrating SKBR3 cell, F-actin and c-erbB-2 molecules are
concentrated in the cell cortex (Fig. 2B
). Using confocal
laser scanning microscopy, we revealed that c-erbB-2 receptors are
localized on the leading edge of a protrusion of a cell starting to
penetrate the 8 µM pore (Fig. 2C
). During penetration, the
c-erbB-2 molecules of the cell wallpaper the inner surface of the pore
in the PET membrane (Fig. 2C
, arrow). At this stage of the
transendothelial invasion process, the F-actin (green), a main molecule
reorganized during cell crawling, was observed within lamellipodia
(Fig. 2C
; ref 24
). As shown in Fig. 2D
,
settlement of the cells on the uncovered side of the membrane was
accompanied by redistribution of the c-erbB-2 receptors to the cellular
cortex colocalizing with F-actin (nonprimary color yellow). Areas of
the cell body still in the process of membrane extension presented a
scattered distribution of the c-erbB-2 molecules both at the attachment
points to the hole and inside the cell (Fig. 2D
, arrow).
F-Actin was organized in a cross-weave structure, which is
characteristic of the traction step in cell crawling (Fig. 2D
; 25).
Phosphorylated c-erbB-2 is localized in protrusions and in parts of
the cell undergoing extreme shape changes during transendothelial
invasion
To clarify whether the observed distribution of c-erbB-2 is
caused by activation or by mere membrane perturbation due to changes in
shape during invasion, we performed experiments in situ
using an antibody specifically recognizing tyrosine phosphorylated
c-erbB-2 (ptyr1248; ref 26
). Tyrosine-phosphorylated c-erbB-2 was found
in cells penetrating the pores in the PET membrane (Fig. 3
, red). In those cells, phosphorylated c-erbB-2 could be assessed at
areas of high membrane extension activity (Fig. 3)
. The leading edges
of those invasive cells are highly positive for phosphorylated
c-erbB-2. EGFR could be costained in the same areas of the cell as the
phosphorylated c-erbB-2 during migration in the
endothelial-extracellular matrix assay (Fig. 3
, green). Moreover, the
most pronounced costaining of phosphorylated c-erbB-2 and EGFR
presented at protrusions of the migrating cells (Fig. 3
, white
nonprimary color). Because c-erbB-2 is 20-fold higher than EGFR in
SKBR3 cells, the red staining for c-erbB-2 is predominantly seen.
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Heterodimerization of c-erbB-2 and EGFR in transendothelial
invasive breast cancer cells
These results support the assumption that the invasive and
motogenic phenotype of the investigated cell lines is related to a
c-erbB-2/EGFR coexpression. This view is further strengthened by the
results from immunoprecipitation and Western blotting experiments with
those cells using an antibody that detects the carboxyl-terminal domain
of EGFR (Ab 1005) and a c-erbB-2-specific antibody decorating the
extracellular domain (Ab-2; Oncogene Science). As shown in Fig. 4
A, the quiescent and EGF-stimulated cell lines SKBR3 and
MHER2 form heterodimers of c-erbB-2 and EGFR. In contrast to the SKBR3
cell, in which the number of c-erbB-2 receptors exceeds that of EGFR,
c-erbB-2 in MDA-Her2 cells is not constitutively activated (Fig. 4B
).
|
Transendothelial invasive cancer cells endocytose activated
c-erbB-2 after induction by EGF
As shown in Fig. 5
a, the c-erbB-2-overexpressing cell line SKBR3 displays
constitutively phosphorylated c-erbB-2 receptors in quiescence that
remain in the plasma membrane. We used EGF as a specific ligand for
induction of EGFR-mediated transphosphorylation of c-erbB-2. This EGF
stimulation led to marked endocytosis of c-erbB-2 surface receptors, as
shown in Fig. 5b
for SKBR3, indicated by cytoplasmic
dot-like fluorescence signals obtained by confocal laser scanning
microscopy. To cover the phosphorylation site, the antibody against
tyrosine-phosphorylated c-erbB-2 was microinjected into SKBR3 cells.
After decorating the anti-phosphotyrosine c-erbB-2 antibody with an
anti-mouse antibody conjugated to LRSC, a rim-like staining in the cell
periphery was observed, which indicates that the c-erbB-2 receptor is
not trafficking out of the plasma membrane when masked with the
anti-phosphotyrosine c-erbB-2 antibody (Fig. 5c
).
Microinjected isotypic antibodies were randomly distributed in the
cytoplasm, providing evidence for a specific binding of the
anti-phosphotyrosine c-erbB-2 antibody (Fig. 5d
).
|
Gelsolin mobilization is inducible in the c-erbB-2 and EGFR
coexpressing transendothelial invasive cancer cells by EGF
Migration during transendothelial invasion requires spatial
changes in the actin component of the cytoskeleton, which is
demonstrated in Fig. 2
. The state of actin assembly is affected by
different actin binding proteins like gelsolin, CapG, and profilin
(21)
. Among those, gelsolin has been demonstrated to
modulate the rate of migration of an individual cell (21)
.
This could be ascribed to the potential of gelsolin to sequester and
nucleate G-actin as well as to cap and to sever F-actin (21
, 22)
. Figure 6
A demonstrates that quiescent SKBR3 cells exhibit a
homogeneous cortical staining for gelsolin. Stimulation of the
c-erbB-2/EGFR signaling pathway by EGF led to mobilization of gelsolin,
shown by a dot-like pattern in the cytoplasm and a decrease in light
emission intensity (Fig. 6B
). This gelsolin release could
also be monitored by flow cytometry, as shown in Fig. 6C
for
the MHER2 cell line. In contrast, the noninvasive MDAwt cell line and
phosphoinositide cleavage-inhibited MHER2 cells did not show any
gelsolin mobilization after EGF treatment. After EGF treatment, the
mean fluorescence of gelsolin staining is reduced in the SKBR3 and
MHER2 cell line by 60 and 30%, respectively. Western blotting
demonstrates that this is not a consequence of gelsolin down-regulation
by EGF (data not shown).
|
The activity of gelsolin in migrating cells is regulated by
phosphoinositides. In fact, we measured in stimulated (100 ng/ml EGF),
migratory, and invasive SKBR3 and MHER2 cells a steep decrease in
phosphoinositol 4,5-diphosphate (PIP2), which dropped to 30–60% of
the initial values within 30 s. After a further 60 s, a rapid
resynthesis of phosphoinositides occurred, which exceeded initial
levels (Fig. 7
). In contrast, we measured in stimulated (100 ng/ml EGF) nonmigratory
and noninvasive MDAwt cells only a PIP2 breakdown, which was not
accompanied by PI resynthesis.
|
Phosphorylated c-erbB-2 and gelsolin are colocalized during
migration through the endothelial-extracellular matrix layer
During the invasion of the SKBR3 (not shown) and MHER2 (Fig. 8
) cells, phosphorylated c-erbB-2 and gelsolin are localized
predominantly around condensed areas where the cells are in contact
with the inner surface of the pore. If the cells move through a narrow
slot, it is obvious they have to reorganize actin fiber structures
spatially and temporally, which can be achieved by the activity of
gelsolin. This is demonstrated by the almost exclusive localization of
gelsolin and F-actin in the moving cells. F-Actin is organized in a
cross-weave structure opposite to the pore, indicating lamellipodia
formation (Fig. 8)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, we have applied breast carcinoma cell lines to a
highly selective in vitro endothelium extracellular matrix
model to mimic the step of invasion of the capillary bed. Increased
expression of c-erbB-2 augmented the invasive capacity of
the cells in this model, as also demonstrated by up-regulation of
c-erbB-2 expression in the former wild-type, low invasive
MDA-MB-468 cell line and down-regulation in the antisense-producing
SKBR3AS cell line. With respect to the SKBR3AS cell line, it has to be
considered that down-regulation of c-erbB-2 by antisense transfection
also leads to growth reduction. However, tumor cells that are growth
inhibited are not necessarily restricted in their migratory behavior,
as shown by Xu et al. using anti-c-erbB-2 antibodies (27)
.
The data of Yu et al. (11)
and Tan et al.
(28)
, who found that the introduction of the human
c-erbB-2 gene into lung and breast cancer cells promotes
migration through extracellular matrix, also support our results.
We demonstrate that the invasive cell line SKBR3 displays
phosphorylated c-erbB-2 at sites of protrusions and extreme shape
changes during transendothelial invasion. The antibody used recognizes
an autophosphorylation site that was found as the essential site for
the full oncogenic potential of neu in fibroblasts (29)
.
Like phosphorylated c-erbB-2, EGFR also stained positive at the
protrusions and areas of extreme shape changes of the moving cell
during transendothelial invasion.
Subsequently, we confirmed a constitutive heterodimerization of
c-erbB-2 and EGFR in SKBR3 and MHER2 cells by Western blotting. This
agrees with results from molecular conformational energy calculations
of c-erbB-2 and EGFR tyrosine kinase, domains predicting that
heterodimers would be preferred over homodimers (13)
.
Thus, if a cell expresses both c-erbB-2 and EGFR, then heterodimer
formation will be favored over homodimerization (14)
.
Down-regulating of cell surface c-erbB-2 and EGFR by intracellular
expression of specific antibodies indicated that c-erbB-2 is also
preferred as a heterodimerization partner for EGFR over both the erbB-3
and erbB-4 receptors (12)
. Using phosphotyrosine-specific
antibodies in a Western blotting experiment, we also showed that
c-erbB-2 undergoes EGF-dependent transphosphorylation by EGFR in SKBR3
and MHER2. No constitutively phosphorylated c-erbB-2 was detected in
MHER2, in contrast to the data from Ennis et al., who reported a low
autocrine expression of EGF/TGF in the MDA-468 wild-type cell line
(30)
. Transphosphorylation induced additional signals that
led to receptor endocytosis, whereas the constitutively activated
c-erbB-2 receptor still remains in the plasma membrane, as shown for
SKBR3 (31)
. This is in accordance with data in the
literature showing that c-erbB-2 is down-regulated after EGF
stimulation in breast and ovarian cancer cell lines
(32
33
34
35)
. Microinjection of a monoclonal antibody directed
against phosphorylated c-erbB-2 into SKBR3 and MHER2 cells inhibited
receptor endocytosis subsequent to EGF stimulation (26)
.
This suggests that the specific phosphorylation at that tyrosine
residue primes the receptors for endocytosis and determines the
receptor traffic; therefore, the time course of receptor signaling.
Furthermore, it could be assumed that c-erbB-2 expression is frequently
accompanied by coexpression of the endogenous EGFR in human cancer cell
lines, as shown in a transgenic mouse model engineered to overexpress
neu, the activated rat analog to c-erbB-2 (36)
. In the
mouse model, overexpression and activation of c-erbB-2 were separable
both temporally and spatially.
Subcellular structural changes needed for transendothelial invasion
require that the spatial and temporal activity of a functional
cytoskeleton, especially actin fibers, has to be modified rather than
disrupted (37
38
39)
. Thus, a question remains: How is
F-actin reorganization linked to c-erbB-2 activation in cells of
transendothelial invasiveness?
The state of actin assembly is affected by different actin-modifying
proteins like gelsolin, CapG, and profilin (21)
. Among
these proteins, gelsolin has been demonstrated to modulate the rate of
migration of an individual cell (22)
. The mobilization and
actin binding of gelsolin in cell migration are dominantly regulated by
phosphoinositides. After EGF stimulation, the transendothelial invasive
cell lines SKBR3 and MHER2 exhibited a time course of phosphoinositide
turnover similar to that observed in platelets undergoing a change in
shape during activation (40)
; we measured a steep decrease
in phosphoinositol 4,5-diphosphate (PIP2) 30 s after EGF
stimulation and a rapid resynthesis of PIP2, which was maintained for
hours. In contrast, the noninvasive MDAwt cells displayed a slow
breakdown and long-lasting lower levels of PIP2.
Subsequent to the c-erbB-2-induced generation of the second messenger
PIP2, membrane-associated gelsolin in SKBR3 and MHER2 cells is
mobilized. The cells thus become more flexible. In addition to confocal
laser scanning microscopy, the gelsolin mobilization could also be
followed by flow cytometry. Since gelsolin is mobilized, it could be
washed out during the procedure of fixation and permeabilization and
the total fluorescence intensity for the FITC dye decorating gelsolin
decreased. The noninvasive MDAwt cell line and PIP2 cleavage inhibited
MHER2, which express high numbers of EGFR molecules, but did
not display any gelsolin mobilization. However, Chen et al. observed a
gelsolin mobilization and an enhanced motogenicity in NR6 fibroblasts
solely transfected for EGFR expression (41)
. The
difference from our model is that they used mesenchymal cells
(fibroblasts), which have a much higher intrinsic capacity of migration
than the epithelium-derived cancer cells. In their experimental
setting, they did not check for c-erbB-2 coexpression, which was
frequently seen in the transgenic mouse model already mentioned
(36)
.
Gelsolin has also been assumed to have a tumor-suppressive function
(42)
. However, a lower expression of gelsolin in the
transendothelial invasive cells than in the noninvasive cells could not
be observed in our study (data not shown). In our view, this could be
taken as additional evidence that separate mechanisms exist for
oncogenesis and metastasis.
During migration through the endothelial-extracellular matrix layer,
SKBR3 and MHER2 cells displayed highly phosphorylated c-erbB-2 and
gelsolin focused at areas of the cell that are locomoting through the
pore. Spatial and temporal reorganization of actin fibers may be
brought about by the activity of gelsolin induced by activated
c-erbB-2. This can be demonstrated by the almost exclusive localization
of gelsolin and F-actin in the moving cells. F-Actin is organized in a
cross-weave structure opposite the pore, probably exhibiting the
traction step in cell crawling (25)
. F-Actin organized in
lamellipodia was also seen in SKBR3 and MHER2 cells settling down on
the uncovered side of the PET membrane (24)
.
At this stage of investigation, we can conclude there is a subtle cross talk between the transmembrane signaling molecule c-erbB-2 and the actin cytoskeleton, including the generation of the important second messenger PIP2 and mobilization of the actin-regulatory protein gelsolin. This molecular cascade could be triggered by a variety of inducers (e.g., EGF) and their ubiquitous precursors in the human body. We believe our study strongly suggests that c-erbB-2, especially in a heterodimer with EGFR, is closely involved in the signaling pathways that induce the alterations in cell morphology required for metastatic cells to leave the capillary bed.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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