The Transcription Factor AP-1 Is Required for EGF-induced Activation of Rho-like GTPases, Cytoskeletal Rearrangements, Motility, and In Vitro Invasion of A431 Cells

Abstract

Human squamous cell carcinomas (SCC) frequently express elevated levels of epidermal growth factor receptor (EGFR). EGFR overexpression in SCC-derived cell lines correlates with their ability to invade in an in vitro invasion assay in response to EGF, whereas benign epidermal cells, which express low levels of EGFR, do not invade. EGF-induced invasion of SCC-derived A431 cells is inhibited by sustained expression of the dominant negative mutant of c-Jun, TAM67, suggesting a role for the transcription factor AP-1 (activator protein-1) in regulating invasion. Significantly, we establish that sustained TAM67 expression inhibits growth factor-induced cell motility and the reorganization of the cytoskeleton and cell-shape changes essential for this process: TAM67 expression inhibits EGF-induced membrane ruffling, lamellipodia formation, cortical actin polymerization and cell rounding. Introduction of a dominant negative mutant of Rac and of the Rho inhibitor C3 transferase into A431 cells indicates that EGF-induced membrane ruffling and lamellipodia formation are regulated by Rac, whereas EGF-induced cortical actin polymerization and cell rounding are controlled by Rho. Constitutively activated mutants of Rac or Rho introduced into A431 or A431 cells expressing TAM67 (TA cells) induce equivalent actin cytoskeletal rearrangements, suggesting that the effector pathways downstream of Rac and Rho required for these responses are unimpaired by sustained TAM67 expression. However, EGF-induced translocation of Rac to the cell membrane, which is associated with its activation, is defective in TA cells. Our data establish a novel link between AP-1 activity and EGFR activation of Rac and Rho, which in turn mediate the actin cytoskeletal rearrangements required for cell motility and invasion.

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The Rockefeller University Press, 0021-9525/98/11/1087/13 $2.00
The Journal of Cell Biology, Volume 143, Number 4, November 16, 1998 1087–1099
http://www.jcb.org 1087
The Transcription Factor AP-1 Is Required for
EGF-induced Activation of Rho-like GTPases,
Cytoskeletal Rearrangements, Motility, and
In Vitro Invasion of A431 Cells
Angeliki Malliri,* Marc Symons,
‡
Robert F. Hennigan,* Adam F.L. Hurlstone,* Richard F. Lamb,*
Tricia Wheeler,* and Bradford W. Ozanne*
*Beatson Institute for Cancer Research, Bearsden, Glasgow, G61 1BD, United Kingdom; and
‡
Onyx Pharmaceuticals,
Richmond, California 94806
Abstract.
Human squamous cell carcinomas (SCC) fre-
quently express elevated levels of epidermal growth
factor receptor (EGFR). EGFR overexpression in
SCC-derived cell lines correlates with their ability to in-
vade in an in vitro invasion assay in response to EGF,
whereas benign epidermal cells, which express low lev-
els of EGFR, do not invade. EGF-induced invasion of
SCC-derived A431 cells is inhibited by sustained ex-
pression of the dominant negative mutant of c-Jun,
TAM67, suggesting a role for the transcription factor
AP-1 (activator protein-1) in regulating invasion. Sig-
nificantly, we establish that sustained TAM67 expres-
sion inhibits growth factor–induced cell motility and
the reorganization of the cytoskeleton and cell-shape
changes essential for this process: TAM67 expression
inhibits EGF-induced membrane ruffling, lamellipodia
formation, cortical actin polymerization and cell round-
ing. Introduction of a dominant negative mutant of Rac
and of the Rho inhibitor C3 transferase into A431 cells
indicates that EGF-induced membrane ruffling and
lamellipodia formation are regulated by Rac, whereas
EGF-induced cortical actin polymerization and cell
rounding are controlled by Rho. Constitutively acti-
vated mutants of Rac or Rho introduced into A431 or
A431 cells expressing TAM67 (TA cells) induce equiv-
alent actin cytoskeletal rearrangements, suggesting that
the effector pathways downstream of Rac and Rho re-
quired for these responses are unimpaired by sustained
TAM67 expression. However, EGF-induced transloca-
tion of Rac to the cell membrane, which is associated
with its activation, is defective in TA cells. Our data es-
tablish a novel link between AP-1 activity and EGFR
activation of Rac and Rho, which in turn mediate the
actin cytoskeletal rearrangements required for cell mo-
tility and invasion.
Key words: AP-1 • Rho-like GTPases • invasion •
motility • EGFR
I
nvasion
, the movement of cells through cellular and
extracellular matrix barriers into neighboring tissue,
distinguishes malignant from benign tumors. Although
a simple pathological distinction, invasion itself is a com-
plex process entailing alterations in cell–cell and cell–extra-
cellular matrix (ECM)
1
interactions, remodelling of the
ECM, reorganization of the cytoskeleton, and increased
cell motility (reviewed in Liotta et al., 1991 and Birch-
meier, 1995). The many facets of invasion imply a require-
ment for coordinated expression and/or activation of mul-
tiple gene products. Previously, we have proposed that the
transcription factor AP-1 (activator protein-1) in oncogen-
Address all correspondence to Prof. B.W. Ozanne, Beatson Institute for
Cancer Research, Switchback Road, Bearsden, Glasgow, G61 1BD, UK.
Tel.: 44 141 942 0855. Fax: 44 141 942 6521. E-mail: b.ozanne@beatson.
gla.ac.uk
Robert F. Hennigan’s present address is Department of Cell Biology,
Neurobiology and Anatomy, University of Cincinnati College of Medi-
cine, Cincinnati, OH 45267.
Richard F. Lamb’s present address is MRC Laboratory of Molecular
and Cellular Biology, University College London, Gower Street, London,
C1E 6BT, UK.
Marc Symon’s present address is Picower Institute for Medical Re-
search, 350 Community Drive, Manhasset, NY 11030.
1.
Abbreviations used in this paper:
AP-1, activator protein-1; CAT,
chloramphenicol acetyl transferase; Col, collagenase; ECM, extracellular
matrix; EGFR, epidermal growth factor receptor; GAP, GTPase-activat-
ing proteins; GDI, guanine nucleotide dissociation inhibitors; GEF, gua-
nine nucleotide exchange factors; HEK, human epidermal keratinocytes;
HGF/SF, hepatocyte growth factor/scatter factor; HPV, human papilloma
virus; JNK, c-Jun NH
2
-terminal kinase; SRF, serum response factor; SCC,
squamous cell carcinomas.
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The Journal of Cell Biology, Volume 143, 1998 1088
ically transformed fibroblasts regulates the expression of
such a multigenic invasion program (Hennigan et al., 1994;
Lamb et al., 1997
a
).
AP-1 encompasses a complex family of transcription
factors composed primarily of heterodimers between Fos
and Jun family proteins (reviewed in Karin et al., 1997).
The prototypes of each family, c-
fos
and c-
jun
, were first
identified in their transduced forms as retroviral onco-
genes, highlighting the important role of AP-1 in transfor-
mation. Subsequently, it has been shown that AP-1 activ-
ity is required for complete transformation by a wide
variety of oncoproteins (Lloyd et al., 1991; Rapp et al.,
1994; Suzuki et al., 1994; Johnson et al., 1996; Kralova et al.,
1998). AP-1 is activated by numerous signal transduction
pathways. Through regulating target gene expression, AP-1
couples short-term signals elicited by extracellular stimuli,
such as growth factors, to long term adaptive changes in
cell phenotype (Curran, 1988). Initial studies in fibroblasts
identified AP-1 as an essential regulator of cell prolifera-
tion (Holt et al., 1986). However, regulating proliferation
is not the sole contribution of AP-1 to malignant transfor-
mation.
A governing role for AP-1 in the regulation of cell
shape, motility, and invasion has also emerged. In
fos
-
transformed fibroblasts there is gross cytoskeletal reorga-
nization, resulting in a diminution of actin stress fibers and
focal contacts, and altered motility (Miller et al., 1984;
Hennigan et al., 1994; Lamb et al., 1997
a
,
b
). Many of the
genes whose expression is either enhanced or repressed in
fos
-transformed fibroblasts, such as Mts-1, CD44, strome-
lysin 1, ezrin, and urokinase-type plasminogen activator
(Hennigan et al., 1994; Miao and Curran, 1994; Jooss and
Muller, 1995; Lamb et al., 1997
a
,
b
), are clearly implicated
in cytoskeletal organization, cell motility, or remodelling
of the ECM. Through the inactivation of AP-1 compo-
nents and ablation of individual AP-1 target gene prod-
ucts, we have demonstrated that AP-1 activity is an essen-
tial requirement for morphological transformation and in
vitro invasion of growth factor-treated and v-
fos
–trans-
formed fibroblasts (Lamb et al., 1997
a
,
b
).
In epidermal cells, a critical role for AP-1 during the
conversion of benign papillomas, induced by Ha-
ras
, into
invasive SCC has been demonstrated using the mouse skin
multistage model of carcinogenesis (Greenhalgh and Yuspa,
1988; Domann et al., 1994
a
; Saez et al., 1995). Other stud-
ies have indicated that the tumorigenicity (Domann et al.,
1994
b
) and in vitro invasiveness (Dong et al., 1997) of
mouse SCC can be suppressed by sustained expression of
a dominant-negative deletion mutant of c-Jun, TAM67,
which inhibits AP-1-mediated transcription (Brown et al.,
1993; Domann et al., 1994
b
; Dong et al., 1997) without af-
fecting proliferation (Domann et al., 1994
b
; Dong et al.,
1997). In human epidermal cells immortalized by expres-
sion of human papilloma virus (HPV) 18, coexpression of
v-
fos
promotes progression to a malignant phenotype (Pei
et al., 1993). Such cells have elevated levels of AP-1 activ-
ity, and expression of TAM67 suppresses their anchorage
independent growth concomitant with loss of AP-1 trans-
activation (Li et al., 1998). The results of the above studies
are consistent with a role for AP-1 in the progression of
benign epidermal cells to invasive SCC. However, the
mechanism by which AP-1 controls a complex process
such as invasion and therefore its precise role in malignant
progression are unknown.
In contrast to the mouse skin carcinogenesis model,
human SCCs rarely possess mutationally activated
RAS
genes (Clark et al., 1993). Instead they display an increase
in the expression of the EGFR (Hendler and Ozanne,
1984; Stanton et al., 1994), first demonstrated for A431
cells (Fabricant et al., 1977). Increased EGFR expression
has diverse consequences: acting through an autocrine
mechanism, it renders the proliferation of A431 cells less
dependent upon an exogenous source of EGF (Fan et al.,
1994). It also enhances EGF-induced motogenic responses
of SCC-derived cell lines compared with human epidermal
keratinocytes (HEKs; McCawley et al., 1997), and may
thus contribute to the invasiveness of SCC cells.
The early responses of A431 cells to EGF stimulation
have been well documented. EGF induces rapid alter-
ations in the organization of the actin microfilament sys-
tem that result in extensive membrane ruffling, lamellipodia
formation, cortical actin polymerization, and cell rounding
(Chinkers et al., 1979; Schlessinger and Geiger, 1981;
Rijken et al., 1991; Peppelenbosch et al., 1993). These pro-
cesses precede and accompany cell movement (reviewed
in Lauffenburger and Horwitz, 1996 and Mitchison and
Cramer, 1996). Actin cytoskeletal rearrangements are reg-
ulated by members of the Rho-like GTPase family in all
cell types surveyed, including epithelial cells (reviewed in
van Aelst and D’Souza-Schorey, 1997 and Hall, 1998). In
epithelial MDCK cells Rac is required for membrane
ruffling and lamellipodia formation during hepatocyte
growth factor/scatter factor (HGF/SF)-induced scattering
(Ridley et al., 1995). Further, Rac and Rho proteins have
been implicated in the invasion of carcinoma cells (Keely
et al., 1997; Shaw et al., 1997; Yoshioka et al., 1998). The
involvement of Rac and Rho in the motility and invasion
of epithelial cells is consistent with findings in fibroblasts
and T-lymphoma cells showing a requirement for these
molecules in
ras
-induced transformation (Qiu et al., 1995
a
,
b
),
as well as in invasion and growth factor-induced chemo-
taxis (Michiels et al., 1995; Anand-Apte et al., 1997). These
studies in combination implicate Rho-like GTPase-induced
cytoskeletal reorganization in the process of invasion.
In this study, we demonstrate that EGF can induce the
in vitro invasion of SCC-derived cells but not benign epi-
dermal keratinocytes. We establish that AP-1 is required
for this response, since sustained
expression of TAM67 in
A431 cells blocks EGF-dependent in vitro invasion. Sig-
nificantly, we find that TAM67 expression inhibits EGF-
induced cell motility and in particular the cytoskeletal
rearrangements and cell shape changes essential for this
process. This occurs because EGF can no longer activate
the small Rho-like GTPases Rac and Rho in TAM67-express-
ing cells, establishing a novel link between EGF signaling,
AP-1 activity, and Rho-like GTPases.
Materials and Methods
Materials
Human recombinant EGF was obtained from R&D Systems (Abingdon,
UK). TRITC- and FITC-labeled phalloidin and propidium iodide were
purchased from Sigma Chemical Co. (Poole, UK), whereas FITC- and
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Malliri et al.
AP-1 Requirement for Rho-like GTPase Regulation
1089
TRITC-labeled anti–mouse immunoglobulin G (IgG) were purchased
from Jackson ImmunoResearch Laboratories (West Grove, PA). Antibod-
ies used to detect TAM67 (anti-c-Jun/AP-1, Ab-1) and EGFR were
obtained from Oncogene Science (Cambridge, UK) and Upstate Biotech-
nology Inc. (Lake Placid, NY), respectively. Other antibodies used were
anti-active MAPK pAb (Promega, Southampton, UK), anti-ERK2 (Trans-
duction Laboratories, Lexington, KY), anti-phosphotyrosine (PY20;
Transduction Laboratories), anti-human Rac (Upstate Biotechnology
Inc.), anti-RhoA (Santa Cruz Biotechnology, Santa Cruz, CA), anti-myc
(9E10; Invitrogen, Leek, The Netherlands) and anti-Met (h-Met C-28;
Santa Cruz Biotechnology). Exoenzyme C3, obtained from Upstate Bio-
technology Inc., was coinjected with FITC-dextran (Molecular Probes,
Inc., Eugene, OR). In vitro invasion chambers (Transwell™; 8-
m
m pore
size filter) were purchased from Costar (Bucks, UK). Reduced growth
factor Matrigel was from Becton Dickinson & Co. (Sparks, MD).
Cell Culture and Transfection Experiments
HEKs, the HPV-16 immortalized nontumorigenic human keratinocyte
cell line TFK104, the human keratinocyte immortalized cell line HaCaT,
the BICR squamous cell carcinoma lines, and MS2 cervical carcinoma
cells were cultured as previously described (Malliri et al., 1996). A431
cells, a human vulvar epidermoid cancer cell line, were routinely main-
tained in DME (GIBCO BRL, Paisley, UK) supplemented with 10%
FCS. All cells were cultured in a humidified atmosphere of 5% CO
2
in
air.
A TAM67 expression construct, pCMVTAM67, expressing a c-Jun de-
letion mutant under the control of a cytomegalovirus promoter (Brown et al.,
1993) was a gift from Dr. Michael Birrer (National Cancer Institute, Be-
thesda, MD) and cotransfected at a 20:1 ratio with pSV2
neo
. Lipo-
fectAMINE (GIBCO BRL) was used to cotransfect subconfluent A431
cells according to the manufacturer’s instructions. Control transfections
were also performed using the pSV2
neo
plasmid only. G418 was added to
the culture medium 72 h after transfection at a final concentration of 500
m
g/ml and resistant colonies were isolated and subcloned by limiting dilu-
tion to generate single-cell subclones. The transfected A431 cells (NA for
cells transfected with pSV2
neo
alone and TA for TAM67-expressing
clones) were maintained in growth medium supplemented with G418.
For the treatment of A431 cells or the transfected NA or TA clones
with EGF, cells plated either on tissue culture plates or on glass coverslips
were allowed to attach overnight in complete medium and then trans-
ferred to serum-free medium for at least 2 d before EGF treatment. Cells
were treated with EGF at a concentration of 10 or 100 ng/ml, or in certain
cases with 2 or 5 ng/ml, with similar results obtained.
AP-1 Transactivation Assay
AP-1 activity in NA and TA cells was assayed using a reporter plasmid
(
2
73 Col-CAT) as previously described (Havarstein et al., 1992). This
plasmid encodes for the chloramphenicol acetyl transferase (CAT) gene
under the control of an AP-1 responsive element present within the colla-
genase (Col) gene promoter. CAT activity is normalized against activity
arising from the
2
63 Col-CAT reporter plasmid, which lacks the AP-1
binding site. 3
3
10
5
cells were seeded in 60-mm plates and transfected
with 4
m
g of either
2
73 Col-CAT or
2
63 Col-CAT using lipofectAMINE.
After transfection was completed, cells were incubated for 24 h in either
serum-containing medium or serum-free medium supplemented with 10
ng/ml EGF. After this time, CAT activity in cell extracts containing equal
amounts of protein was determined by standard techniques (Gorman et
al., 1982).
In Vitro Invasion Assay
An inverse invasion assay in serum-free conditions was performed and
quantitated as previously described (Hennigan et al., 1994; Lamb et al.,
1997
a
), using growth factor depleted Matrigel-coated polycarbonate
chambers (Transwell™ 8-
m
m pore size filters). In brief, in this assay cells
were first allowed to attach to the underside of a Transwell™, and then
chemoattracted across the 8-
m
m pore size filter and into the Matrigel
above. Cells were fixed and stained with propidium iodide and nuclei at
regular 10-
m
m confocal z-sections from the bottom of the filter visualized
using a MRC 600 confocal illumination unit (Bio-Rad Laboratories, Her-
cules, CA) attached to a Nikon Diaphot inverted microscope. Images of
labeled cell nuclei at each height were processed as tagged-image file for-
mat (TIFF) images, and pixels quantitated using a computer program de-
veloped and described before (Hennigan et al., 1994).
Cells at and above 20
m
m from the bottom of the filter (i.e., in the
Matrigel layer) have no cellular contact with the filter and are thus consid-
ered to have invaded the matrix (data not shown). Percentage chemotaxis
is the ratio of positive pixels at and above the top of the filter to the total
combined number of pixels above and below the filter, whereas percent-
age invasion is the ratio of pixels at and above 20
m
m from the bottom of
the filter to the total number of pixels above and below the filter. For ad-
dition of EGF to the assay, human recombinant EGF at a concentration
of 10 ng/ml was added above the Matrigel layer in serum-free DME.
Transwells™ were placed in the wells of conventional 24-well tissue cul-
ture plates containing serum-free DME.
Western Blot Analysis
For all Western blots, cells were washed twice in cold PBS. With the ex-
ception of the EGFR Western blot, cells were then lysed for 30 min on ice
in 50 mM Hepes, pH 7.0, 250 mM NaCl, 0.5% Nonidet P-40, 5 mM
EDTA, 50 mM NaF, 200
m
M sodium orthovanadate, 2 mM benzamidine,
50 mM
b
-glycerophosphate, 10 mM sodium pyrophosphate, 1 mM PMSF,
10
m
g/ml aprotinin, 10
m
g/ml leupeptin, and 10
m
g/ml soybean trypsin in-
hibitor. For the EGFR Western blot, cells were lysed in 50 mM Hepes, pH
7.5, 150 mM NaCl, 1.5 mM MgCl
2
, 1 mM EGTA, 10% glycerol, 1% Triton
X-100, 100 mM NaF, 10 mM sodium pyrophosphate, 1 mM sodium ortho-
vanadate, 50 mM
b
-glycerolphosphate, 2 mM PMSF, 10
m
g/ml aprotinin,
and 10
m
g/ml leupeptin. Lysates were centrifuged for 15 min at 14,000 rpm
at 4
8
C. Protein concentrations were determined by bicinchoninic acid as-
say (Sigma Chemical Co., St. Louis, MO). 50–80
m
g of extracted protein
were fractionated by SDS-PAGE and transferred electrophoretically to
polyvinylidene difluoride membranes (Immobilon P; Millipore Corp.,
Bedford, MA). Immunoblots were blocked and probed with antibodies
against TAM67, EGFR, active MAPK, ERK2, c-Met, phosphotyrosine,
Rac, and RhoA according to protocols supplied by the manufacturers.
The Western blots were developed with the enhanced chemiluminescence
Western blotting system (Nycomed Amersham, Buckinghamshire, UK).
Blots were then visualized by exposure to Kodak X-Omat film for various
times.
Scattering Assays
The stimulatory effect of EGF on cell motility was measured for A431,
NA, and TA clones. Cells were seeded at a density of 2
3
10
3
cells/well on
6-well plates. Cells were allowed to attach and when they had formed
small colonies were transferred to serum-free medium for
z
2 d. Colonies
were then treated with EGF (2, 5, or 10 ng/ml) for 2 d. Colonies were pho-
tographed before and two days after the addition of growth factor using
phase-contrast optics.
Wound-healing Assays
A431, NA, or TA cells were plated in 60-mm tissue culture dishes at 2
3
10
6
cells/plate, cultured overnight in complete medium, and when a con-
fluent monolayer was formed transferred to serum-free medium for 2 d.
The confluent monolayers were wounded using a disposable plastic pi-
pette tip and washed with serum-free medium. Photographs were taken
just after wounding and 48 h later using phase-contrast optics.
Identical wounding assays were performed with monolayers of NA and
TA cells grown on glass coverslips, in which case cells were fixed and
stained for F-actin, as below, 24 h after wounding.
Digital Time-lapse Microscopy
Cells plated on 60-mm tissue culture dishes at a density of 10
5
cells/plate
were left to attach overnight in complete medium. Cells were then trans-
ferred to serum-free medium for a minimum of 2 d. Before digital time-
lapsing, medium was replaced with Hepes-buffered serum-free DME and
cells filmed using an MRC 600 Nikon Diaphot confocal laser microscope
at 37
8
C for 5 min before and 45 min after the addition of EGF. Phase sig-
nals at 30-s intervals were collected with a COMOS program (Bio-Rad
Laboratories) and processed as TIFF images. Several similar fields of
A431, NA, and TA cells were filmed with identical results.
Microinjection
Cells for microinjection were seeded on glass coverslips, left to attach
overnight in complete medium, and then transferred to serum-free me-
dium for two days before microinjection. C3 transferase was microinjected
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The Journal of Cell Biology, Volume 143, 1998 1090
in the cytoplasm of NA13 cells at a concentration of 200
m
g/ml, together
with FITC-Dextran as an inert marker protein to identify injected cells.
C3 transferase at this concentration inhibited serum induced stress fiber
formation in 208F fibroblasts (data not shown). C3 was microinjected over
a 10-min period and then cells were returned to the incubator for a further
15 min before treating them with EGF for either 5 or 15 min before fixa-
tion. The myc-tagged constructs pEXV14RhoA, pEXV12Rac1 encoding
constitutively active forms of RhoA and Rac1, respectively (Qui et al.,
1995
a
,
b
), were microinjected into the nucleus of NA13 and TA37 cells.
12–18 h later cells were fixed and double stained for the myc-epitope
and actin filaments using an anti-myc mAb (9E10) and phalloidin, re-
spectively. NA13 cells were similarly microinjected with a plasmid,
pEXVN17Rac1, encoding a dominant-negative mutant form of Rac1
tagged with the myc-epitope (Qui et al., 1995
b
). Microinjected cells left to
express the plasmid 12-18 h were treated with EGF for either 5 or 15 min
and then fixed and stained as above. Microinjection was performed using
a microscope (Axiovert 135m; Zeiss, Inc., Thornwood, NY) attached to an
Eppendorf Microinjection Unit (Microinjector model 5242, Micromanipu-
lator model 5170, and Heat Controller model 3700). During microinjec-
tion cells were maintained at 37
8
C and before microinjection medium was
replaced with Hepes-buffered serum-free DME. Microinjected plasmids
and C3 were diluted in microinjection buffer (5 mM potassium glutamate/
150 mM KCl).
Immunofluorescence
For immunofluorescence cells were fixed in 4% formaldehyde for 15 min
at room temperature and washed several times in PBS containing 0.1%
Triton X-100. For visualization of filamentous actin, fixed cells were
stained with 500 ng/ml phalloidin for 10 min at room temperature in PBS
containing 0.1% Triton X-100. For myc-epitope staining of the microin-
jected cells blocking of nonspecific antibody binding was performed with
PBS containing 0.1% Tween-20 and 5% skimmed milk (Marvel) for 45
min at room temperature. Blocked slides were incubated for 1 h at room
temperature in blocking buffer with 1:20 dilution of anti-myc antibody
(9E10), washed and then incubated for 45 min at room temperature in
fresh blocking buffer containing anti–mouse IgG. Stained slides were
washed and mounted (Vectashield; Vector Laboratories, Inc., Burlin-
game, CA). For visualization of endogenous Rac, cells were blocked in
BSA and stained according to the protocol supplied by the manufacturer.
Control staining with an isotype matched primary antibody indicated that
Rac immunocytochemical staining was specific for the antibody used
(data not shown). All slides were viewed on the laser confocal microscope.
Fluorescence images were obtained with conventional FITC and TRITC
excitation settings on the laser confocal microscope described above and
processed as TIFF images.
Images perpendicular to the substratum (y-z projections) of the cells
were constructed, using laser scan confocal microscopy on phalloidin
stained cells, allowing visualization of cell height.
Results
SCC-derived Cell Lines Invade in Response to EGF
SCC-derived cell lines express elevated levels of EGFR
compared with normal human keratinocytes in culture
(Hendler and Ozanne, 1984; Stanton et al., 1994).
We used
a quantitative in vitro invasion assay (Hennigan et al.,
1994; Lamb et al., 1997
a
) to test whether EGFR overex-
pression correlates with increased invasion potential. For
our studies we used a number of well characterized SCC-
derived cell lines (Edington et al., 1995; Malliri et al., 1996)
for which the levels of EGFR expression are known (Stan-
ton et al., 1994). We also included normal HEKs, HPV16
E6 and E7 immortalized HEKs (TFK104), and benign im-
mortal keratinocytes (HaCaT) to determine whether inva-
siveness is an exclusive property of malignant epithelial
cells. Unlike the SCC-derived cell lines, none of the benign
keratinocyte cell lines overexpress EGFR (Stanton et al.,
1994). Further, none of the benign epidermal cells invaded
in this assay, even in the presence of EGF (Fig. 1
a
). In
contrast, whereas none of the SCC-derived cell lines tested
invaded spontaneously, they all invaded after EGF treat-
ment (Fig. 1
a
). A431 cells are an example of the SCC-
derived cell lines shown in Fig. 1
a
. Without EGF no A431
cells migrated to the top of the filter; on addition of EGF
cells migrated to a distance of 50
m
m from the bottom of
the filter (Fig. 1
b
). Invasion of A431 cells was dependent
upon the concentration of EGF added to the top of the
Matrigel and was maximal for 10 ng/ml (data not shown).
Invasion was abrogated both by anti-EGFR antibodies
demonstrated to inhibit EGFR signaling (Modjtahedi et
al., 1993) and by CGP52411 (a gift from Dr. N. Lydon,
Ciba Geigy Pharmaceuticals, Basel, Switzerland) that in-
hibits EGFR tyrosine kinase activity (Buchdunger et al.,
1994; data not shown). We conclude that invasion of SCC-
derived cells lines correlates with EGFR overexpression
and is dependent on EGFR signaling.
Figure 1. Invasion response of normal epidermal and SCC-
derived cell lines. (a) Quantitative analysis of invasion of primary
keratinocytes (HEK), immortalized keratinocytes (TFK104 and
HaCaT), and various SCC-derived cell lines in response to 10 ng/ml
EGF. Invasion assay results were quantitated as described else-
where (Hennigan et al., 1994; Lamb et al., 1997a) with a Bio-Rad
program (Comos) and represent the average from at least three
independent experiments. (b) A431 invasion in response to EGF.
Shown are confocal images of propidium iodide–stained cell nu-
clei at the bottom of the filter (0 mm), top of the filter (10 mm),
and at various heights as indicated through the Matrigel in the
absence (top) or presence (bottom) of 10 ng/ml EGF.
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Malliri et al.
AP-1 Requirement for Rho-like GTPase Regulation
1091
Invasion of A431 Cells Is Inhibited by Expression of the
Dominant Negative Mutant of c-Jun, TAM67
Previously, we have demonstrated that invasion of v-
fos
-
transformed or EGF-treated fibroblasts requires func-
tional AP-1 (Lamb et al., 1997
a
). To determine if AP-1 ac-
tivity is required for EGF-dependent in vitro invasion of
human SCC-derived cell lines, we generated A431 trans-
fectants constitutively expressing the c-Jun deletion mu-
tant, TAM67. A431 cells were selected because their
response to EGF treatment is well characterized and be-
cause they demonstrate a marked invasion response to
EGF. TAM67 has been clearly demonstrated to nullify
AP-1 transcriptional activity (Brown et al., 1993; Domann
et al., 1994
b
; Dong et al., 1997; Li et al., 1998). G418-resis-
tant A431 subclones were obtained and tested by Western
blot analysis for expression of TAM67. Three A431
TAM67-expressing clones, TA5, TA36, and TA37, and
two nonexpressing G418-resistant clones, NA13 and NA15,
were used most extensively in subsequent experiments
(Fig. 2
a
). Sustained TAM67 expression was found to in-
hibit basal and EGF-induced AP-1 transcriptional transac-
tivation using a collagenase promoter-CAT reporter con-
struct (Fig. 2
b
). As was previously demonstrated for
malignant mouse epidermal cell lines (Domann et al.,
1994
b
; Dong et al., 1997), introduction of TAM67 did not
alter the growth rates of expressing clones (data not
shown).
The in vitro invasion assay was used to determine
whether sustained expression of TAM67 altered the abil-
ity of A431 cells to respond to EGF. 14 TA clones and 12
NA clones were examined for their invasive response to
EGF treatment (10 ng/ml). Whereas all the NA clones re-
tained their invasive response to EGF, none of the TA
clones invaded the Matrigel in response to EGF. Quantita-
tion of the invasion of two representative NA clones and
three representative TA clones is shown in Fig. 2
c
. Confo-
cal images of cell nuclei from the invasion assay are also
shown for one NA and one TA clone in Fig. 2
d
. It was ev-
ident from the analysis of the in vitro invasion assay that
chemotaxis itself of TA cells was impaired, as cells failed
to migrate even to the top of the micropore filter (Fig. 2
c
).
All the SCC-derived cell lines that invade in vitro in re-
sponse to EGF, including A431 cells, have high levels of
EGFR (Stanton et al., 1994). We tested whether the im-
paired invasiveness of TA cells resulted from decreased
EGFR expression and/or signaling activity. Quantitation
of the level of EGFR expression for A431 and NA and TA
clones by Western blotting (Fig. 3
a
) or by ligand binding
assays (data not shown) revealed that the level of EGFR
expression was equivalent in TA, NA, and A431 cells. Fur-
thermore, EGF-induced EGFR autophosphorylation was
equivalent in A431, NA, and TA cells, as determined by
Western blot analysis using an anti-phoshotyrosine anti-
body (Fig. 3
b
), and by an EGFR in vitro
kinase assay
(data not shown). The ability of EGF to stimulate phos-
phorylation of MAPK was also equivalent in TA, NA, and
A431 cells (Fig. 3
c
). Thus, TAM67 expression inhibits
chemotaxis and invasion of A431 cells without directly in-
terfering with EGFR expression, ligand activation of its ty-
rosine kinase activity, or its ability to activate MAPK.
TAM67 Inhibits EGF- and HGF/SF-stimulated Colony
Scattering as Well as Wound Closure
The greatly reduced chemotactic response of TA cells to
EGF stimulation suggested that they might display im-
paired motility in response to EGF. A431, NA, and TA
cells normally grow as packed colonies. Colonies of A431
cells respond to HGF/SF ligation of its receptor, c-Met, by
Figure 2. Expression of TAM67 in-
hibits AP-1 transactivation and inva-
sion of A431 cells. (a) Western blot
analysis of A431 cells, two NA
clones, and three TA clones using a
polyclonal antibody against c-Jun to
reveal expression of TAM67. (b)
Measurement of AP-1 directed CAT
expression in NA and TA cells as
described in Materials and Methods.
Columns show the average for three
independent experiments. (c) Quan-
titative analysis of chemotaxis and
invasion of A431, NA, and TA cells
in response to EGF as described in
Materials and Methods and else-
where (Hennigan et al., 1994; Lamb
et al., 1997a). Columns show the av-
erage for four independent experi-
ments. (d) Confocal images of pro-
pidium iodide stained cell nuclei of
NA13 and TA37 cells at the bottom
(0 mm) and at a distance 30 mm from
the bottom of the filter (i.e., 20 mm
into the Matrigel) after EGF stimu-
lation.
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The Journal of Cell Biology, Volume 143, 1998 1092
scattering, which requires the disruption of cell–cell junc-
tions and an increase in cell motility (Tajima et al., 1992).
Like the EGFR, c-Met is a ligand stimulated protein ty-
rosine kinase. We found that EGF, in addition to HGF/SF,
induced scattering in A431 cells, as had previously been
demonstrated for SCC-derived cells HSC-1, which also
overexpress EGFR (Fujii et al., 1996). To assess whether
inhibition of AP-1 activity directly inhibits motility of
A431 cells, we tested both EGF and HGF/SF for colony
scattering activity on A431 cells, two NA clones, and three
TA clones. EGF efficiently induced scattering of A431 and
NA colonies within 48 h, but did not induce scattering of
TA colonies (Fig. 4 a). Treatment of A431 or NA cells
with the EGFR inhibitor CGP52411 inhibited EGF-stimu-
lated colony scattering (data not shown). Similar results
were obtained with HGF/SF treatment even though A431,
NA, and TA cells all expressed equivalent levels of c-Met
(data not shown). These results suggest that TAM67 inhib-
its growth factor-stimulated cell–cell disruption and/or cell
motility.
Monolayers of cells respond to wounding by migrating
into the wound to effect closure. Closure of wounds of
z0.75 mm in monolayers of A431 or NA cells occurred
within 48 h (Fig. 4 b) by a process that was independent of
mitomycin C treatment (at a concentration 0.15 mg/ml that
inhibits DNA synthesis by 90%) and therefore of cell pro-
liferation (data not shown). Wounding itself was a potent
stimulus for cell migration. In contrast to A431 and NA
cells, wounded monolayers of TA cells did not migrate sig-
nificantly into the wound, failing to close it over a 48-h pe-
riod (Fig. 4 b).
We noted that in EGF-induced colony scattering, scat-
tered cells appeared rounded with pronounced lamellipo-
Figure 3. (a) NA and TA clones express similar levels of EGFR.
Western blot analysis of EGFR in A431 cells, two NA, and three
TA clones using a sheep anti–human EGFR polyclonal antibody.
(b) EGF-induced EGFR autophosphorylation is equivalent in
A431, NA, and TA clones. The top panel represents a Western
blot for phoshotyrosine, using an anti-phosphotyrosine–specific
mouse mAb, after EGF treatment of A431 cells, NA, and TA
clones. The bottom panel represents a similar Western blot
probed with sheep polyclonal anti-EGFR antiserum to show
equivalent loading as well as similar levels of EGFR expression
between the different clones. (c) EGF stimulates phosphoryla-
tion of MAPK in A431, NA, and TA clones. The top panel repre-
sents MAPK phosphorylation detected using an antibody specific
for the phosphorylated forms of MAPK. The bottom panel repre-
sents a similar Western blot probed with an anti-ERK2–specific
antibody to demonstrate equal loading.
Figure 4. Expression of TAM67 inhibits motility of A431 cells.
(a) Colonies of cells were treated with EGF as described in Mate-
rials and Methods. Representative A431, NA, and TA colonies,
photographed using a phase contrast microscope, are shown be-
fore and 48 h after the addition of 10 ng/ml EGF. (EGF was also
used at concentrations of 2 and 5 ng/ml with similar results ob-
tained.) Insets show higher magnification images of two different
motile cells. (b) Wounding assays for NA13 and TA37 cells.
Wounds were created in monolayers of NA13 or TA37 cells as
described in Materials and Methods and photographed immedi-
ately and 48 hours later. (c) Wounds created in monolayers of
NA13 and TA37 cells grown on glass coverslips were fixed and
stained for F-actin with phalloidin 24 after wounding. Photo-
graphs show cells at the edge of the wound. Results for one of
four independent experiments are shown. Bars: (a, c) 20 mm; (b)
100 mm.
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Malliri et al. AP-1 Requirement for Rho-like GTPase Regulation 1093
dia (Fig. 4, a and insets). Time-lapse digital microscopy of
EGF-treated A431 cells revealed that the rounded cells
were motile and extended lamellipodia in the direction of
movement (data not shown). In the wound healing assays,
cells that had moved into the wound were also rounded.
Rounded cells displayed an increase in F-actin that was
polarized in its distribution with a higher concentration to-
wards the wound (Fig. 4 c). In contrast, TA cells, which did
not move in response to EGF or wounding, did not round
(Fig. 4 c). These results suggest that cell rounding is part of
the motogenic response of A431 cells.
TAM67 Inhibits EGF-stimulated Rearrangements of
the Actin Cytoskeleton and Cell Rounding/Contraction
EGFR activation leads to rapid and pronounced mem-
brane ruffling in A431 cells, which is followed by sig-
nificant cortical actin polymerization (Chinkers, 1979;
Schlessinger and Geiger, 1981; Rijken et al., 1991; Peppe-
lenbosch et al., 1993). No increase in actin stress fibers, a
common consequence of growth factor stimulation of fi-
broblasts and other epithelial cells (van Aelst and D’Souza
Schorey, 1997; Hall 1998), has been noted for A431 cells.
As a consequence of cortical actin polymerization, cells in-
crease in height as they contract and become rounded.
Pretreatment of A431 or NA cells with the EGFR inhibi-
tor CGP52411 prevented EGF-induced actin cytoskeletal
rearrangements and cell rounding (data not shown) indi-
cating that EGF-stimulated EGFR tyrosine kinase activity
was required to initiate these rapid changes.
We investigated whether EGFR activation results in ac-
tin cytoskeletal rearrangements in TA cells, culminating in
cell rounding. Serum-starved NA and TA cells displayed
low levels of F-actin as judged by phalloidin staining (Fig.
5, a and b). EGF treatment of NA cells resulted in mem-
brane ruffles (Fig. 5 c) and lamellipodial extensions (Fig. 5
c, insert) within 5 min, which was followed by a dramatic
increase in polymerized cortical actin by 15 min (Fig. 5 e).
No increase in actin stress fibers was detected at 5 or 15
min after EGF treatment (Fig. 5, c and e). When TA cells
were treated with EGF, membrane ruffling, lamellipo-
dia formation, and cortical actin polymerization were all
greatly reduced (Fig. 5, d and f). The cortical actin poly-
merization occurring in NA cells 15 min after EGF treat-
ment was associated with an increase in cell height. The
bottom panels of Fig. 5, a, b, e, and f show confocal micros-
copy optical sections made perpendicular to the substra-
tum through the cells at the position of the colony indi-
cated by the arrow. As judged by comparing these bottom
panels, 15 min of EGF treatment resulted in a significant
Figure 5. EGF treatment leads to lamellipodia formation, mem-
brane ruffling, and cortical actin polymerization in NA but not
TA cells. Confocal micrographs of rhodamine-phalloidin stained
NA13 and TA37 cells before (a and b), 5 min (c and d), and 15
min (e and f) after the addition of EGF. The main micrograph in
c shows a z-section towards the top of the cells to reveal mem-
brane ruffles (arrowheads), whereas the insert shows an en-
hanced z-section at the base of the cells to reveal lamellipodia
(arrowhead). No z-section through TA cells revealed ruffles or
lamellipodia (d) even when enhanced to the same degree as the
inset in c (d, inset). Bottom panels of a, b, e, and f show optical
sections made perpendicular to the substratum through the cells
of the colonies above, at the position of the colony indicated by
the arrow. (Cells were treated with 100 ng/ml EGF. Similar re-
sults were also obtained with 10 ng/ml.) Photomicrographs shown
are representative of four independent experiments. Bar, 10 mm.
Figure 6. A431, NA13, but not TA36 and TA37 cells contract/
round up in response to EGF. The response of cells to EGF was
recorded by time-lapse digital microscopy for 5 min before and
40 min after the addition of EGF as described in Materials and
Methods. (Cells were treated with 100 ng/ml EGF. Similar results
were also obtained with 10 ng/ml.) Photomicrographs shown are
representative of four independent experiments. Bar, 20 mm.
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The Journal of Cell Biology, Volume 143, 1998 1094
increase in cell height in NA cells, which was much re-
duced in TA cells. The presence of membrane ruffles,
lamellipodia, and polymerized cortical actin was tested in
A431 cells and clones NA13, NA15, TA5, TA36, and
TA37 with similar results as shown in Fig. 5 for clones
NA13 and TA37. 40 min after treatment with EGF, the
great majority of A431 and NA cells became rounded/con-
tracted, whereas TA cells did not (Fig. 6). EGF-induced
rounding was tested in eight NA and eight TA clones with
results similar to those shown in Fig. 6 for A431 cells and
clones NA13, TA37, and TA36. We conclude that sus-
tained expression of TAM67 inhibits EGF-induced cy-
toskeletal rearrangements and cell rounding.
Rac and Rho Mediate EGF-induced Cytoskeletal
Rearrangements in A431 Cells. EGF Fails to Activate
Rho-like GTPases in TA Cells
To study the involvement of Rho-like GTPases in EGF-
induced cytoskeletal changes, Rac1 was selectively inhib-
ited by microinjection of NA13 cells with a plasmid encod-
ing a myc-epitope-tagged dominant negative mutant of
Rac1 (DN-Rac) and Rho by microinjection of Clostridium
botulinum exoenzyme C3 transferase. After an overnight
period, following microinjection of DN-Rac, or 15 min af-
ter the microinjection of C3 transferase, cells were treated
with EGF for 5 or 15 min and stained for F-actin with
phalloidin. Expression of DN-Rac inhibited membrane
ruffles and lamellipodia formation (Fig. 7, a and b), indi-
cating that Rac activation is required for the formation of
these membrane protrusions in EGF-treated NA13 cells.
No accumulation of polymerized cortical actin nor cell
rounding was detected in the C3 transferase-microinjected
NA13 cells after 15 min of EGF treatment (Fig. 7, e and f),
although membrane ruffles were observable after 5 min
EGF treatment (data not shown), indicating that C3 trans-
ferase was not inhibiting Rac activity. This observation
demonstrates that Rho activity is required for EGF-induced
cortical actin polymerization and cell rounding. Further-
more, treatment of NA13 cells microinjected with DN-Rac
with EGF for 15 min blocked cortical actin polymerization
(Fig. 7, c and d). This suggests that Rac activation is up-
stream of Rho activation in the EGF-dependent pathway
regulating cortical actin polymerization in A431 cells.
The inability of EGF to induce membrane ruffles, lamel-
lipodia, and cortical F-actin in TA cells suggests that
EGFR signaling to the actin cytoskeleton is compromised
in these cells. This could be due to a failure to activate Rac
Figure 7. Inactivation of Rac and Rho in NA13 cells inhibits
EGF-induced actin rearrangements. Confocal micrographs of
cells microinjected with an expression construct encoding a myc-
tagged version of RacN17 (a–d) or with C3 transferase and FITC-
dextran (e and f), treated with EGF for either 5 min (a and b) or
15 min (c–f), and costained either for myc and phalloidin (cells in
a–d) or for phalloidin alone (e and f). Microinjected cells were
detected either by myc-tag specific mAb (9E10)-staining (b and d)
or the presence of FITC-dextran (f). Arrowheads in a point to
lamellipodia and membrane ruffles in noninjected cells, and in
c and e indicate noninjected cells with cortical F-actin. A repre-
sentative field of cells for at least three independent experiments
is shown for each treatment. Bar, 10 mm.
Figure 8. Activated forms of Rac and Rho function equally in
NA13 and TA37 cells. Confocal micrographs of cells microin-
j
ected with expression constructs encoding myc-tagged versions
of either V12Rac (a–d) or V14Rho (e–h) and stained for the myc-
tag using a myc specific mAb (9E10; to detect the injected cells)
and phalloidin (for the visualization of polymerized actin). Ar-
rowheads in a and c indicate colocalization of Rac and F-actin at
sites of cell–cell contacts. A representative field of cells for at
least three independent experiments is shown for each treatment.
Bar, 10 mm.
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Malliri et al. AP-1 Requirement for Rho-like GTPase Regulation 1095
and Rho, the absence or functional inactivity of certain
downstream effectors of Rac and Rho, or a combination of
both. To determine whether the downstream effectors of
Rac and Rho responsible for the EGF-induced cytoskele-
tal rearrangements are present and functional in TA cells,
plasmids directing the expression of myc-epitope-tagged
activated forms of Rac1 and RhoA (RacV12 and RhoV14)
were introduced by microinjection into NA13 and TA37
cells. Expression of RacV12 resulted in the appearance of
F-actin at the site of cell to cell contacts, where RacV12
also accumulated (Fig. 8, a–d), as has also been reported
for MDCK cells (Ridley et al., 1995; Hordijk et al., 1997;
Takaishi et al., 1997). Expression of RhoV14 resulted in an
increase in F-actin around the cell cortex and rounded
morphology resembling that which occurred after EGF
treatment (Fig. 8, e–h). It also induced a low but detect-
able increase in actin stress fibers, which did not occur af-
ter EGF treatment (data not shown). These results indi-
cate that the main cytoskeletal rearrangement in A431
cells mediated by Rho is the polymerization of cortical ac-
tin rather than the formation of stress fibers. Most signifi-
cantly, however, actin structures formed in both NA13
cells and TA37 cells after microinjection of either RacV12
or RhoV14 were equivalent (compare Fig. 8 a with c and e
with g). These results indicate that the downstream ef-
fectors of Rac and Rho required for the specific EGF-
induced actin cytoskeletal and morphological changes are
functional in TA cells, which suggests that EGF activation
of Rac and Rho is compromised in TA cells.
No assay is presently available to directly measure the
activation of Rho. However, in mouse dermal fibroblasts
Rac has been shown to be recruited from the cytoplasm to
the plasma membrane upon growth factor stimulation
concomitant with its activation (Azuma et al., 1998). In se-
rum-deprived NA13 and TA37 cells Rac as detected by
immunofluorescence was not found associated with the
plasma membrane (Fig. 9 A, a and b). Upon EGF stimula-
tion though Rac was detected in the plasma membrane of
NA13 but not TA37 cells (compare Fig. 9 A, c and e with d
and f), indicating that EGF stimulation does not activate
Rac in TA cells.
One explanation for the absence of actin cytoskeleton
rearrangements after EGF treatment in TA cells could be
due to decreased expression of Rac and Rho in TA cells.
This possibility was excluded by the demonstration that
the levels of Rac and Rho protein in extracts from A431,
NA13, NA15, TA36, and TA37 cells, as determined by
Western blot analysis using antibodies specific for Rac and
Rho, were equivalent (Fig. 9 B).
Discussion
Through investigating the regulation of human SCC inva-
sion, we have made several interesting findings that estab-
lish a novel link between EGF signaling, AP-1, and the
regulation of cell motility. First, in vitro invasion of SCC-
derived cells is dependent upon EGF stimulation, and this
response to EGF does not occur in benign epidermal cells.
Second, expression of the c-Jun deletion mutant, TAM67,
inhibits EGF-induced cytoskeletal rearrangements neces-
sary for membrane ruffling, lamellipodia formation, cell
rounding, and ultimately motility and invasion. Third,
TAM67 expression inhibits EGF activation of Rho-like
GTPases, the critical regulators of these events.
We have shown that invasion in response to EGF distin-
guishes SCC-derived cells from benign epidermal cells, in-
cluding those that are immortal. An obvious distinction
between the malignant and benign epidermal cells is their
level of expression of EGFR, which suggests that overex-
pression of growth factor receptors could be an important
step in the emergence of the invasive phenotype. This is
supported by the finding that whereas colon-adenoma–
derived cells do not invade in vitro in response to EGF,
malignant variants of these cells that overexpress the
EGFR do (Brunton et al., 1997). However, EGFR overex-
pression may not be sufficient to render benign cells re-
sponsive to EGF-induced invasion, since loss of invasion
suppressor genes also appears to be critical for the inva-
sive phenotype of cancers (Liotta et al., 1991; Birchmeier
et al., 1995).
EGF-induced in vitro invasion of the SCC-derived cell
Figure 9. (A) Rac translocates to membrane ruffles in NA13 but
not TA37 cells after 5 min treatment with EGF. Confocal micro-
graphs are shown for NA13 and TA37 cells stained using a mouse
anti-Rac mAb. (a and b) NA13 and TA37 cells untreated; (c and e)
NA13 cells treated with 100 ng/ml EGF for 5 min; (d and f) TA37
cells treated with 100 ng/ml EGF for 5 min. A representative field
of cells for at least three independent experiments is shown. (B)
Western blot analysis demonstrating equivalent levels of expres-
sion of Rac and Rho in A431, NA, and TA cells. Bar, 10 mm.
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The Journal of Cell Biology, Volume 143, 1998 1096
line A431 is dependent upon AP-1 activity since sustained
expression of TAM67 in these cells inhibited this response,
concomitant with suppression of AP-1 transactivation. In
this study, we establish that a major role of AP-1 in facili-
tating invasion of carcinoma cells is its ability to couple
growth factor signaling to cell motility. We demonstrate
that TAM67-mediated inhibition of AP-1 results in both
impaired chemotaxis and scattering of A431 cells in re-
sponse to EGF.
The early responses of A431 cells to EGF, which include
formation of membrane ruffles and lamellipodia, cortical
actin polymerization, and cell body retraction (rounding),
are all processes assumed to occur repeatedly during the
long-term response of cell migration (Lauffenburger and
Horwitz, 1996; Mitchinson and Cramer, 1996). Sustained
TAM67 expression inhibited not only the long-term mo-
tile response of A431 cells to EGF, but also these early re-
sponses. We contend that the failure of EGF to induce cy-
toskeletal and morphological changes in TA cells is in
large part responsible for their impaired motile response.
Further, our findings imply that continued expression of
one or more AP-1 target genes is required in order to
transduce an EGF signal into a motogenic response. This
hypothesis is at least consistent with the findings of Ridley
et al. (1995) who also demonstrated a requirement for de
novo gene expression during HGF/SF-induced motility of
MDCK cells.
In this study, we establish that EGF-induced actin cy-
toskeletal rearrangements and morphological changes in
A431 cells are dependent upon activation of Rac and Rho,
as documented for many other cell types (van Aelst and
D’Souza-Schorey, 1997; Hall, 1998). Whereas Rac is re-
quired for EGF-induced membrane ruffling and lamelli-
podia formation in A431 cells, Rho regulates cortical actin
polymerization and cell rounding. Further, our findings
that (a) dominant-negative Rac1 inhibits cortical actin po-
lymerization and cell rounding, as well as lamellipodia for-
mation and membrane ruffling; (b) C3 transferase inhibi-
tion of Rho inhibits only EGF induced cortical actin
polymerization and cell rounding; and (c) activated RhoA
by itself is sufficient to induce cortical actin polymeriza-
tion only are consistent with the proposed hierarchy sug-
gested for Swiss 3T3 cells, where growth factor stimulation
of Rho-mediated events are dependent upon Rac activa-
tion (Nobes and Hall, 1995). However, unlike the situation
in fibroblasts (Nobes and Hall, 1995), activated Rac1 by it-
self appears insufficient to activate Rho in A431 cells.
In fibroblasts and epithelial MDCK cells, Rho-mediated
actin polymerization results in an increase in stress fibers
(Ridley and Hall, 1992; Ridley et al., 1995). However, in
A431 cells expression of RhoV14 or activation of Rho by
EGF stimulation results largely in cortical actin polymer-
ization and cell rounding, but not stress fiber formation. A
similar response to Rho activation occurs in neuroblas-
toma-derived cells (Kranenburg et al., 1997; van Leeuwen
et al., 1997). This suggests that the type of actin reorgani-
zation mediated by Rho in response to growth factor sig-
naling is cell type and perhaps stimulus specific.
As already alluded to, we believe that Rho-mediated
rounding observed after EGF treatment of A431 cells is an
important component of their motogenic response. We
have noted in both EGF-induced cell scattering and wound
healing experiments that the motile cells are rounded. In
the wound healing experiments the leading edge cells
round up, break away from the monolayer, and migrate
into the wound. During EGF-induced scattering cell
rounding precedes and is maintained during movement.
Time-lapse digital microscopy reveals that the round mo-
tile cells extend lamellipodia in the direction of movement.
This mode of movement is not restricted to A431 cells and
reflects a more common form of epidermal cell motility,
since normal HEK cells stimulated to move also migrate
as round cells (Owens, D., unpublished data).
From the absence of actin cytoskeletal rearrangements
in EGF-treated TA cells, coupled with loss of motility and
invasiveness, we postulated that expression of TAM67
might be compromising signaling pathways involving Rac
and Rho. In principle, this block could occur either up-
stream of Rac and Rho preventing their activation, at the
level of expression of Rac and Rho, or downstream of Rac
and Rho by reducing the activity of effector molecules, or
a combination of all of these. Our results are most consis-
tent with the first explanation. Rac activation is associated
with membrane translocation (Azuma et al., 1998), and
whereas EGF induces the translocation of Rac to the
plasma membrane in NA cells, it does not in TA cells. No
significant alterations in the level of expression of Rac or
Rho were detected in TA cells. Furthermore, expression
of constitutively active forms of Rac1 and RhoA induced
indistinguishable cytoskeletal effects in TA and NA cells,
indicating that at least a subset of the effector pathways
downstream of Rac and Rho are intact in both cell types.
However, constitutively active forms of Rac and Rho
alone did not induce A431 or NA cells to move within a
48-h time period (data not shown), indicating that neither
molecule in the absence of EGF signaling is sufficient to
induce cell motility. Expression of dominant negative Rac
within these cells prevented EGF-stimulated cytoskeletal
rearrangements, cell rounding, and motility (Fig. 5 and
data not shown), indicating that Rac activation is required
for all these responses. To the extent that motility is re-
quired for invasion, we contend that Rac would also be re-
quired for invasion.
On the basis of the above analysis, we anticipate that
possible candidates for AP-1 target genes required for
EGF-induced motogenic responses include the multiple
known upstream regulators of Rho-like GTPase activity
(van Aelst and D’Souza-Schorey, 1997; Hall, 1998), and
potentially other as yet unidentified regulators. Two as-
pects appear to be involved in growth factor regulation of
Rho-like GTPase activity: one is the recruitment of these
molecules from the cytosol to the membrane, the other is
guanine nucleotide exchange. However, the precise se-
quence of these events has yet to be determined. A num-
ber of guanine nucleotide exchange factors (GEFs) have
been identified that catalyze guanine nucleotide exchange.
GTPase-activating proteins (GAPs) serve to inhibit Rho-
like GTPases by accelerating the conversion of GTP-
bound forms to their inactive GDP-bound forms. Guanine
nucleotide dissociation inhibitors (GDIs) sequester Rho-
like GTPases in the cytosol and also inhibit GEFs from
stimulating the exchange of GDP for GTP. Members of
the ERM family of proteins, which include ezrin, radixin,
and moesin, can function as activators of Rho-like GTP-
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Malliri et al. AP-1 Requirement for Rho-like GTPase Regulation 1097
ases by mediating their release from complexes with
GDIs. Release from GDIs coincides with membrane
translocation (reviewed in Bretscher et al., 1997; Sasaki et
al., 1998). Interestingly, the expression of both ezrin and
CD44, a membrane-spanning ECM receptor protein that
interacts with ERM proteins (reviewed in Tsukita et al.,
1997; Bretscher et al., 1996), are increased in v-fos–trans-
formed fibroblasts, where they colocalize in microvilli and
at the leading edge of the extending pseudopod (Jooss and
Muller, 1995; Lamb et al., 1997a,b).
Alterations in the expression of other AP-1–regulated
genes required for cell motility and invasion, but not in-
volved with regulation of Rho-like GTPase activity, may
also occur in TA cells. These genes could include pro-
teases that degrade ECM (Hennigan et al., 1994; Miao and
Curran, 1994; Tremble et al., 1995), as well as proteins that
mediate cell–cell adhesion (Reichmann et al., 1992; Fialka
et al., 1996).
It has been demonstrated in mammalian cells that Rho-
like GTPases act upstream of signaling cascades that stim-
ulate expression and activity of AP-1 components, as well
as functioning to alter cell shape and motility. Rho-like
GTPases have been reported to activate the c-Jun NH2-
terminal kinase (JNK; Coso et al., 1995; Minden et al.,
1995; Teramoto et al., 1996) and are also required for stim-
ulation of the serum response factor (SRF; Hill et al.,
1995). Our results suggest the possibility of a positive feed-
back connection between AP-1 function and activation of
Rho-like GTPases that could serve to amplify a motogenic
and invasion stimulus.
Dorsal closure during Drosophila embryogenesis pro-
vides another instance where AP-1 and Rho-like GTPases
function coordinately to regulate cytoskeletal changes and
movement of epithelial cells (reviewed in Martin-Blanco,
1997). During dorsal closure, Rac in the leading most mi-
grating cells activates AP-1 via the JNK signaling pathway,
whereas AP-1 directs the expression of genes such as deca-
pentaplegic, Dpp (a TGF-b homologue). Dpp in turn elic-
its cytoskeletal changes in more laterally positioned epi-
thelial cells. Cell shape changes in leading edge cells,
whereas dependent upon AP-1 activity, are independent
of Dpp signaling (Riesgo-Escovar and Hafen, 1997a). In
more lateral epithelial cells, Dpp signaling still appears to
require DFos function (Riesgo-Escovar and Hafen, 1997b).
This has lead to the suggestion that certain cytoskeletal el-
ements or their regulators are direct effectors of AP-1 sig-
naling during dorsal closure. Based on our results, we sug-
gest that this connection also exists in vertebrate epithelial
cells.
In conclusion, we propose that sustained expression of
TAM67 functions as an inhibitor of a multigenic invasion
program through suppressing the activity of AP-1. A sub-
set of AP-1 target genes appear to provide molecular
bridges between growth factor stimulation and activation
of Rac and Rho, which in turn regulate morphological and
motile responses (Fig. 10). In this respect, AP-1 target
genes could include upstream activators of Rac and Rho
(Fig. 10, X), but equally might include inhibitors of these
molecules (Fig. 10, Y). The effect of TAM67 on down-
stream signaling from the EGFR appears specific to the
Rho-like GTPase cascade, since the ability of EGFR to ac-
tivate MAPK is unimpaired in TA cells (Fig. 10). Our find-
ings strengthen the hypothesis that AP-1 activity is essen-
tial for the invasion of malignant human epithelial cells.
We would like to thank Prof. Wyke and Drs. Parkinson, Frame, Gillespie,
and Stapleton, of the Beatson Institute for Cancer Research for critical
reading of this manuscript. We are indebted to Dr. Iain Morgan for help
with the CAT assays to measure AP-1 activity in our cell lines. We would
also like to thank Dr. M. Birrer for providing us with the TAM67 plasmid,
Dr. N. Fusenig for the HaCat cells, Dr. K. Vousden for the TFK104 cells,
and Peter McHardy for assistance with microscopy.
We gratefully acknowledge the Cancer Research Campaign for their fi-
nancial support.
Received for publication 19 June 1998 and in revised form 16 September
1998.
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