Epidermal Growth Factor Protects Epithelial Cells against
Fas-induced Apoptosis
REQUIREMENT FOR Akt ACTIVATION*
Spencer
Gibson
§,
Shine
Tu
,
Ryan
Oyer
,
Steven M.
Anderson¶, and
Gary L.
Johnson
From the
Program in Molecular Signal Transduction,
Division of Basic Sciences, National Jewish Medical and Research
Center and the Departments of ¶ Pathology and
Pharmacology,
University of Colorado Medical School, Denver, Colorado 80206
 |
ABSTRACT |
Chemotherapeutic drugs that damage DNA kill tumor
cells, in part, by inducing the expression of a death receptor such as
Fas or its ligand, FasL. Here, we demonstrate that epidermal growth factor (EGF) stimulation of T47D breast adenocarcinoma and embryonic kidney epithelial (HEK293) cells protects these cells from Fas-induced apoptosis. EGF stimulation of epithelial cells also inhibited Fas-induced caspase activation and the proteolysis of signaling proteins downstream of the EGF receptor, Cbl and Akt/protein kinase B
(Akt). EGF stimulation of Akt kinase activity blocked Fas-induced apoptosis. Expression of activated Akt in MCF-7 breast adenocarcinoma cells was sufficient to block Fas-mediated apoptosis. Inhibition of
EGF-stimulated extracellular signal-regulated kinase (ERK) activity did
not affect EGF protection from Fas-mediated apoptosis. The findings
indicate that EGF receptor stimulation of epithelial cells has a
significant survival function against death receptor-induced apoptosis
mediated by Akt.
 |
INTRODUCTION |
Expression of epidermal growth factor
(EGF)1 receptor family
members (ErbB1-4) is observed in many human tumors, particularly of
the breast, ovary, and lung (1-4). Overexpression of EGF receptors in
these tumors is correlated with poor prognosis for responsiveness to
radiation and chemotherapy (1, 5-7). The proliferative response and
resistance to chemotherapy afforded by EGF receptor activation in these
tumors may allow for the rapid outgrowth of drug-resistant tumor cells
(3, 6). Clinically, there is interest in developing strategies to test
if a given tumor will respond to a given treatment of radiation and
combinations of chemotherapy. This testing could allow the optimization
of drug combinations and treatment modifiers to enhance tumor cell growth.
In the past several years, it has been realized that many
chemotherapeutic drugs induce apoptosis of tumor cells (8). Studies with human tumor cells of diverse tissue origin indicate that apoptosis
is the common end point for drug-induced cell death. This is true for
the spectrum of DNA-damaging agents, anti-metabolites, and microtubule
toxins (8-10). Cumulatively, these findings have led to the
realization that cells don't actually die from overwhelming DNA damage
or injury per se, but rather, specific regulatory pathways are activated that induce apoptosis. Thus, a key in successful anti-tumor therapy is to enhance the susceptibility of the tumor cell
to undergo apoptosis.
An emerging theme from studies defining the mechanisms of action of
chemotherapeutic drugs is the involvement of death receptors, particularly Fas and its ligand, FasL, in drug-induced apoptosis. Fas
activation by FasL activates a caspase cascade leading to apoptosis
(11-14). Treatment of different tumor cell types with DNA-damaging
drugs has been shown to induce the expression of Fas and/or FasL (12).
Blocking Fas activation in these cells inhibits drug-induced apoptosis
of the tumor cells (12, 13, 15). These findings have demonstrated a
signal transduction response from DNA damage to the transcriptional
regulation of death receptors and their ligands. In this report we show
that activation of EGF receptors in tumor cells of epithelial origin protects against Fas-induced apoptosis.
 |
MATERIALS AND METHODS |
Cell Culture--
Cells were maintained in a humidified 7.0%
CO2 environment in Dulbecco's modified medium supplemented
with 100 units/ml penicillin, 100 µg/ml streptomycin (Life
Technologies, Inc.). Media for T47D, MCF7, and HEK293 cells was
supplemented with 20% bovine calf serum, 10% fetal bovine serum, and
10% bovine calf serum (Life Technologies, Inc.), respectively. MCF7
cells expressing vector Akt K-M and Akt-myr protein were under
selection with 0.5 mg/ml G418 (Life Technologies, Inc.). Each cell line
was tested for the presence of EGF receptors and the Fas receptor.
EGF and Fas Stimulation--
Cells (1-2 × 106) were suspended in 1 ml of Dulbecco's modified medium
medium-containing serum to minimize incubation volumes. The cells were
incubated with or without 1 µg/ml EGF (Biochemical Technology) for
1 h at 37 °C. After the incubation, 1 µg/ml activating anti-Fas antibody (Upstate Biotechnology Inc.) was added, and cells
were incubated at room temperature for 10 min. Two ml of Dulbecco's
modified medium-containing serum was added to the cells, and the
mixture was placed in tissue culture plates. Adherent cells were
incubated at 37 °C in a 7.0% CO2 incubator for 24 or 48 h where indicated. Wortmannin (100 nM, Sigma) and
PD098059 (50 µM, Parke-Davis) was added at the same time
as EGF where indicated. Similar results were obtained when EGF and
activating anti-Fas antibody were added directly to adherent cells.
Akt Kinase Assay--
Cells were lysed in Akt lysis buffer (10 mM K3PO4, pH 7.4, 1 mM
EDTA, pH 8.0, 5 mM EGTA, 10 mM
MgCl2, 20 mM
-glycerophosphate, 0.5 µM sodium vanadate, 2 mM dithiothreitol, 40 ng/ml phenylmethylsulfonyl fluoride, 0.1% Nonidet P-40, 40 µg/ml
aprotinin, and 40 µg/ml leupeptin). Cellular debris was removed by
centrifugation at 8,000 × g for 5 min. Protein
concentration was determined by a Bradford assay using bovine serum
albumin as a standard. Four hundred µg of cell lysates was
immunoprecipitated with 2 µg/ml of an anti-Akt1 antibody (Santa Cruz
Biotechnology Inc.) for 1 h at 4 °C with agitation. This was
followed by the addition of 15 µl of a 1:1 slurry of protein
A-Sepharose beads (Sigma) and incubated at 4 °C for 1 h. The
beads were then washed twice in 1 ml of lysis buffer and twice in Akt
wash buffer (20 mM Tris, pH 7.5, 10 mM MgCl2, 0.1 mg/ml bovine serum albumin, 1 mM
dithiothreitol, 1 µg/ml protein kinase A inhibitor peptide).
Thirty-five µl of the last wash was left in the tube and mixed with
25 µl of Akt reaction mix (Akt wash buffer, 0.2 mM ATP,
0.2 µg/ml cross-tide peptide (GRPRTSSFAEG), 0.2 µCi/µl
[
32P]ATP) and incubated for 20 min at 30 °C. The
reaction was stopped with 10 µl of 0.5 M EDTA and spotted
on P81 Whatman paper. The samples were washed three times for 5 min
each in 75 mM phosphoric acid and air-dried. The samples
were placed in scintillation vials and radioactivity quantitated.
ERK Kinase Assay--
Cells were lysed in TX-100 lysis buffer
(70 mM
-glycerophosphate, 1 mM EGTA, 100 µM sodium vanadate, 1 mM dithiothreitol, 2 mM MgCl2, 0.5% Triton X-100, 20 µg/ml
aprotinin). Lysate were treated as described in Akt kinase assay. ERK2
was incubated with 2 µg/ml anti-ERK2 antibody (Santa Cruz
Biotechnology Inc.) for 1 h as described for the Akt kinase assay.
The beads were washed twice with 1 ml of lysis buffer and twice with 1 ml of lysis buffer without Triton X-100. Thirty-five µl of the last
wash was left in the tube and mixed with 20 µl of ERK reaction mix
(50 mM
-glycerophosphate, 100 µM sodium
vanadate, 20 mM MgCl2, 200 µM
ATP, 0.5 µCi/µl [
32P]ATP, 400 µM
epidermal growth factor receptor peptide 662-681, 100 µg/µl IP-20,
2 mM EGTA) incubated for 20 min at 30 °C. The reaction
was stopped with 10 µl of 500 mM EDTA and spotted on to
P81 Whatman paper. The samples were washed four times with 75 mM phosphoric acid, air-dried, and counted in a
counter.
Immunoblots--
Cells were lysed in Nonidet P-40 lysis buffer
(50 mM HEPES, pH 7.25, 150 mM NaCl, 50 µM ZnCl2, 50 µM NaF, 2 mM EDTA,
1 mM sodium vanadate, 1.0% Nonidet P-40, 2 mM
phenylmethylsulfonyl fluoride). Cell debris was removed by
centrifugation at 8,000 × g for 5 min, and protein
concentration was determined by a Bradford assay. Two hundred to 400 µg of cell lysate protein was subject to SDS-polyacrylamide gel
electrophoresis and transferred to nitrocellulose membranes. The
membranes were blocked in Tris-buffered saline, 5% milk. Blots were
performed as described in Widmann et al. (19).
Caspase Assay--
Cells were lysed in 50 mM
Tris-HCl, pH 7.4, 1 mM EDTA, 10 mM EGTA, and
100 µg/ml digitonin. Sixty mg of lysate protein was incubated with 5 µM DEVD-7-amido-4-methylcoumarin (Bachem) in 1 ml of 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, and 10 mM EGTA for 30 min at 37 °C. Fluorescence was then
monitored using an excitation wavelength of 380 nM and an
emission wavelength of 460 nM as per manufacture
instructions. The caspase activity was measured as arbitrary
fluorescence units and converted to fold increases over basal caspase
activity in untreated cells. Fluorescence of substrate alone was
subtracted in each case. To validate that we were measuring caspase
activity, multiple inhibitors of caspase activity such as CrmA were
used that reduced DEVD cleavage as measured by fluorescence to
background levels.
Measurement of Apoptosis--
Cells (1-2 × 106) were resuspended in 100 µl of media by gentle
vortexing and 2 µl of acridine orange (100 µg/ml), and 100 µg/ml
ethidium bromide in phosphate-buffered saline was added. Ten µl was
removed and placed on a microscope slide, and a coverslip was applied
over the 10 µl. The slide was viewed on a fluorescence microscope
using a fluorescein filter set for the detection of condensed DNA in
apoptotic cells. The condensed DNA was determined by intense local
staining of DNA in the nucleus compared with the diffuse staining of
the DNA in normal cells. The percentage of apoptotic cells was
determined from cells containing normal DNA staining compared with
cells with condensed DNA. Apoptosis was verified by propidium iodide
staining for DNA fragmentation and morphological changes consistent
with apoptotic cells.
 |
RESULTS |
EGF Stimulation of Epithelial Cells Inhibits Fas-induced
Apoptosis--
EGF stimulation of T47D breast adenocarcinoma and human
embryonic kidney epithelial (HEK293) cells significantly diminished the
percentage of apoptotic cells in response to activation of Fas relative
to control cells not exposed to EGF (Fig.
1A). Stimulation of Fas in
T47D cells resulted in 57 and 74% of the cells to be apoptotic at 24 and 48 h, respectively. Forty-eight h after activation of Fas in
HEK293 cells, 66% of the cell population was apoptotic. EGF
stimulation before Fas ligation reduced the apoptotic response 48 h later to 38% and 32% in T47D and HEK293 cells, respectively. Similar results were observed with activation of Fas by an activating anti-Fas antibody (Fig. 1A) or soluble FasL (not shown).
Quantitation of apoptosis by acridine orange or propidium iodide
staining gave similar results. The findings show that in two different
epithelial cell types, EGF stimulation reduces the ability of Fas
activation to induce cell death by apoptosis.

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Fig. 1.
EGF treatment of T47D and HEK293 cells
reduces Fas-induced apoptosis. A, T47D cells
(i) and HEK293 cells (ii) were preincubated with
or without 1 µg/ml EGF for 60 min. Where indicated, 1 µg/ml
activating anti-Fas antibody was added to the culture medium. After the
indicated times, the apoptotic index for each treatment was quantitated
by acridine orange staining. Error bars represent the S.D.
of three separate experiments. B, cells were stimulated with
EGF and activating anti-Fas antibody as above. At 48 h, caspase
activity was determined. Caspase activity is represented as fold
increase over basal levels. Experiments were done in triplicate with a
variability of less than 15%. C, T47D and HEK293 cells were
treated for 24 h as described in A. Cells were lysed,
proteins were resolved by SDS-polyacrylamide gel electrophoresis, and
immunoblots were performed with antiserums against Cbl, Akt, or MEK1.
Experiments shown in B and C were done three
times, with similar results.
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|
Fas-mediated Caspase Activation Is Inhibited by EGF Stimulation of
Epithelial Cells--
Caspases are the cysteine proteases that when
activated cleave proteins at defined aspartic acid-containing
recognition sequence motifs (16, 17). The cumulative action resulting
from caspase cleavage events commit cells to apoptosis (16-18).
Measurement of caspase activity using a fluorescent DEVD peptide
substrate for caspase 3-like proteases demonstrated Fas stimulates
caspase activity 3- and 2-fold, respectively, in T47D and HEK293 cells (Fig. 1B). Prior treatment of T47D and HEK293 cells with EGF
effectively inhibits caspase activation in response to Fas ligation in
both cell types. This result indicates that EGF stimulates signal
pathways that inhibit Fas activation of caspases. The decreased caspase activation would effectively inhibit Fas-mediated apoptosis.
We recently demonstrated that Fas stimulated
caspase-dependent cleavage of several proteins involved in
EGF signaling including Akt and Cbl (19). Consistent with the
inhibition of caspase activation by EGF (Fig. 1B), EGF
stimulation of T47D and HEK293 cells inhibited the proteolysis of Akt
and Cbl in response to Fas ligation (Fig. 1C). Protection of
Cbl and Akt from caspase-catalyzed cleavage was maintained even after
48 h of Fas activation in the continued presence of EGF (not
shown). The expression of mitogen-activated protein kinase kinase 1 (MEK1), which is not a caspase substrate (19), is not changed during
Fas activation in cells. Thus, the cleavage by caspases of specific
signaling proteins, but not every signaling protein, is orchestrated
during the apoptotic response initiated by Fas ligation. EGF
stimulation of T47D and HEK293 cells inhibits the
caspase-dependent cleavage of proteins in response to
ligation of the death receptor, Fas.
Activation of Akt Protects It from Fas-induced
Degradation--
Akt is activated by EGF stimulation in several cell
types (20, 21). Indeed, EGF stimulation of T47D cells activates Akt (Fig. 2A). Akt activation also
has been shown to protect cells from stress-induced apoptosis (22-24).
The caspase-dependent cleavage of Akt would be expected to
prevent this anti-apoptotic response. Therefore, we examined if EGF
stimulation of T47D cells sustains Akt activation in Fas-activated
cells. The activation of Akt was determined with an antibody specific
for the phosphorylated serine 473 of Akt. Serine 473 is located in the
kinase domain of Akt, and its phosphorylation is required to indicate
Akt activation (23, 24). Increased phosphorylation of Akt was detected
within 5 min after EGF stimulation (Fig. 2B). Twenty-four h
after Fas ligation, Akt was still activated in EGF-treated cells (Fig.
2B). Furthermore, a second challenge with EGF increased the
phosphorylated activated form of Akt in cells exposed to Fas. In
contrast, Fas activation in cells that were not prestimulated with EGF
resulted in no measurable Akt protein (Fig. 2B). These
results show that EGF stimulation of T47D cells not only activates Akt
but also protects it from degradation by caspases in Fas-stimulated
cells.

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Fig. 2.
Activation of Akt after EGF and Fas
stimulation of cells. A, T47D cells were treated with 1 µg/ml EGF in 10% serum for the times indicated. The cells were
lysed, and Akt was immunoprecipitated and assayed by in
vitro kinase assay using cross-tide peptide as the substrate as
described under "Material and Methods." The increase in Akt kinase
activity was determined by fold increase over basal levels.
B, T47D cells incubated for 24 h with or without 1 µg/ml EGF and with or without 1 µg/ml activating anti-Fas antibody.
As indicated, cells were stimulated an additional 5 min with 1 µg/ml
EGF. The cells were lysed and Western blotted using an antibody
directed against the phosphoserine 473 of Akt. The blots were stripped
and reprobed with an anti-Akt antibody. The results are representative
of three independent experiments.
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Akt but Not ERK Activation Is Required for EGF-mediated Protection
from Apoptosis--
The ability of specific signal transduction
pathways to have anti-apoptotic regulatory properties is variable in
different cell types (25). For example, both Akt and the ERK
mitogen-activated protein kinase pathway have been shown to be
anti-apoptotic in different cell types and in response to different
stress stimuli (23, 25-28). Fig. 3 shows
that EGF stimulation of T47D cells activates both Akt and ERK
activities. The time course in response to EGF stimulation of ERK
activation (Fig. 3A) is similar to that for Akt (Fig.
2A), although ERK activation peaked at 10 min, whereas Akt
activation peaked at 30 min. Activation of ERK in response to EGF
stimulation is effectively inhibited by incubation of T47D cells with
the Parke-Davis compound, PD098059 (Fig. 3B). This compound
stimulates the degradation of MEK1 and -2, the mitogen-activated protein kinase kinase in the ERK pathway (29). The activation of Akt is
regulated by phosphorylated phosphatidylinositols that are formed as
products of phosphatidylinositol 3-kinase-catalyzed reactions. The
pleckstrin homology domain of Akt binds to the plasma
membrane-associated phosphatidylinositol (3, 4, 5) trisphosphate or
phosphatidylinositol (3, 4) bisphosphate, resulting in the
translocation of Akt from the cytosol to the plasma membrane.
Membrane-associated Akt is phosphorylated by phosphoinositide-dependent
kinase, leading to its activation (20, 22). As predicted, inhibition of
phosphatidylinositol 3-kinase by wortmannin inhibits EGF-stimulated Akt
activity in T47D cells (20, 30, 31) (Fig. 3C).

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Fig. 3.
Both PD098059 and wortmannin inhibited
EGF-induced ERK and Akt activation respectively. A,
time course of ERK activation in response to EGF was determined as
described under "Materials and Methods." B, T47D cells
in 10% serum were preincubated with or without 50 µM
PD098059 (PD) for 30 min. Cells were then challenged with 1 µg/ml EGF for 10 min. Cells were lysed, and ERK activity was assayed
as in A. C, T47D cells in 10% serum were
preincubated for 10 min with or without 100 nM wortmannin
and then challenged with or without 1 µg/ml EGF. After 30 min, the
cells were lysed and assayed for Akt activity. The results are
represented as a fold increase over basal levels and representative at
least three independent experiments.
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|
Fig. 4 shows that wortmannin but not
PD098059 inhibits the ability of EGF to protect T47D cells from
Fas-induced apoptosis. The anti-apoptotic function of EGF is completely
abrogated by pretreatment with wortmannin (Fig. 4A). In
contrast, inhibition of ERK activation has no effect on the ability of
EGF to protect against Fas-induced apoptosis. The ability of wortmannin
to inhibit the anti-apoptotic function of EGF is mirrored in the
regulation of caspase activity (Fig. 4B). The suppression of
Fas-induced caspase activity by EGF stimulation of T47D cells is
completely inhibited by wortmannin treatment (Fig. 4B). The
effect of wortmannin on caspase activation is also demonstrated by
wortmannin blocking the ability of EGF to protect Cbl and Akt from
caspase-mediated degradation (Fig. 4C).

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Fig. 4.
Wortmannin, but not PD098059,
blocks EGF inhibition of Fasinduced apoptosis. A, T47D
cells were incubated with or without 1 µg/ml EGF, 1 µg/ml
activating anti-Fas antibody, 100 nM wortmannin, and 50 µM PD098059 (PD) in the indicated combinations
for 24 h. The apoptotic index of cells for each combination of
treatments was determined. * represents a p value <0.05
using Student's t test for the difference between Fas
activation versus Fas + EGF and Fas + EGF + PD098059
treatments. The experiment was repeated three separate times.
B, cells treated as in A were lysed and assayed
for caspase activity as described under "Material and Methods."
Caspase activity was measured as fold increase over basal levels, and
the experiments were repeated three times with variability to less than
15%. C, cells treated as in B were lysed,
resolved by SDS-polyacrylamide gel electrophoresis, and immunoblotted
using anti-Cbl or anti-Akt antiserum.
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|
The ability of Akt to protect epithelial cells from Fas-induced
apoptosis is also observed in MCF-7 breast adenocarcinoma cells (Fig.
5). As shown in T47D cells, EGF
stimulation suppresses Fas-induced apoptosis (Fig. 5A).
Stable expression of a constitutively activated Akt in MCF-7 cells
lowers the basal apoptotic cell index relative to control MCF-7 cells
(Fig. 5B). In contrast, a kinase-inactive form of Akt (Akt
K-M) has no effect on the basal apoptotic index of MCF-7 cells.
Stimulation of Fas results in a significant apoptotic response in both
Akt K-M and control MCF-7 cells. In contrast, expression of the
myristoylated constitutively active form of Akt (Akt-myr) strongly
protected MCF-7 cells from Fas-induced apoptosis. Thus, activation of
Akt seems sufficient to inhibit Fas-induced apoptosis.

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Fig. 5.
Akt activation protects MCF-7 human breast
adenocarcinoma cells from Fas-induced apoptosis. A,
MCF-7 cells in 10% serum were incubated with or without 1 µg/ml EGF,
1 µg/ml activating anti-Fas antibody, or the combination of EGF + Fas
antibody for 48 h. The apoptotic index of cells in each treatment
was determined by staining with acridine orange. Error bars
represent the S.D. from three experiments. B, stable
transfectants of MCF-7 cells having the expression vector without an
insert or expressing kinase-inactive Akt (Akt K-M) or constitutively
activated Akt (Akt-myr) were incubated with or without 1 µg/ml
Fas-activating antibody for 48 h. Apoptosis was determined by
acridine orange staining. The results are from three experiments, with
error bars showing the S.D. for each condition.
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|
 |
DISCUSSION |
We have shown that EGF stimulation of three different cell lines
protects epithelial cells from Fas-induced apoptosis. The activation of
Akt appears to be both required and sufficient for the anti-apoptotic
function of EGF (Fig. 6). EGF also has
been shown to protect rat prostatic epithelial cells and cultured fetal hepatocytes from tumor necrosis factor
-induced apoptosis (32, 33).
In other cell types the potential anti-apoptotic function of EGF is
less apparent. In fibroblasts, EGF is not able to significantly protect
against UV-B-induced apoptosis unless highly overexpressed (21, 34). In
contrast, insulin-like growth factor 1 was able to block UV-B-induced
apoptosis (21). Similarly, EGF was not able to block tumor necrosis
factor
-induced apoptosis in adipocytes (35). The differential
ability of EGF to regulate Akt activity in these cell types may explain
these differences. Thus, it appears that epithelial cells and
fibroblasts have different abilities for EGF to signal anti-apoptotic
responses. It is not clear whether this is because of receptor
concentration or which EGF receptor family members are present.

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Fig. 6.
Model of EGF protection of Fas-induced
apoptosis. EGF receptor (EGFR) activation leads to
activation of Raf1 and the ERK pathway. PD098059 compound inhibits the
kinase activity of MEK1/2, thereby blocking EGF-induced activation of
ERK. Phosphatidylinositol 3-kinase (PI3K) is also activated
by stimulation of the EGF receptor, leading to Akt activation.
Wortmannin inhibits phosphatidylinositol 3-kinase activity, thereby
blocking Akt activation. Akt effectively inhibits Fas-induced caspase
activity and apoptosis.
|
|
It should be noted that even though the ERK pathway did not have an
anti-apoptotic function in the three epithelial cell lines we tested,
ERK has been shown to have significant survival functions in other cell
types. For example, in L929 fibrosarcoma cells and human neutrophils,
the ERK pathway prevents tumor necrosis factor
and UV-induced
apoptosis, respectively (36-38). Thus, different cell types may use
either Akt or ERK signaling pathways for survival in response to
different pro-apoptotic stimuli. Recently, it was shown that Akt
phosphorylates both Bad and caspase 9, inhibiting their pro-apoptotic
activity. The phosphorylation of these proteins appears to be a
mechanism for the anti-apoptotic function for Akt (18, 39, 40). The
mechanism by which ERK can function to protect specific cell types from
apoptosis is not currently defined.
Our finding that in human breast epithelial tumor cells, EGF protects
against apoptosis is particularly relevant to understanding the
function of EGF receptors in breast cancer. Human breast tumor cells
express EGF receptors that contribute to their growth and survival (1,
3, 6). Many breast cancer cells express multiple members of the ErbB
family including ErbB1 (EGF receptor) and ErbB2 (Neu) (1, 6). In
addition, many breast cancers also express either EGF or transforming
growth factor
and are thus able to activate EGF receptors in the
tumor cells via autocrine and paracrine mechanisms (41). The
stimulation of the Akt-signaling pathway in breast tumors would also be
predicted to provide a survival function during the transformation
process. Overexpression of ErbB2 receptor by transfection of MDA-MB-435
breast cancer cells resulted in protection of these cells from
taxol-induced apoptosis, indicating that EGF receptor stimulation can
also confer drug resistance (42). Our results indicate that EGF
stimulation of breast cancer cells protects them from Fas-induced
apoptosis by a mechanism involving the activation of Akt.
It is important to note that EGF treatment of epithelial cells
significantly reduced but did not eliminate Fas-induced apoptosis. The
incomplete protection could be because of the fact that we were
activating endogenous EGF receptor family members and endogenous Akt.
In HEK293, MCF7, and T47D cells, ErbB family members and Akt are
expressed at modest levels. Overexpression of an activated form of Akt
or overexpression of EGF receptors effectively blocks apoptosis (42).
Our study is the first report showing that EGF can significantly
protect cells against Fas-induced apoptosis without increasing the
expression of EGF-signaling components by transfection.
The finding that EGF protects breast cancer cells from apoptosis is
consistent with the recent observations using the anti-Neu (ErbB2)
antibody, Herceptin (4). Herceptin blocks EGF signaling and induces
apoptosis of breast cancer cells and is now in use clinically in the
treatment of breast cancer. It is possible that inhibition of the Akt
stimulatory pathway will further sensitize cells to Herceptin-induced
apoptosis (4). Combined with chemotherapy, multimodal treatment would
effectively induce apoptosis in part by making the tumor cells more
sensitive to Fas activation. The ability to define the anti-apoptotic
potential of signaling pathways like that for Akt in different tumor
types should allow enhanced efficiency of treatments involving drugs
like Herceptin, DNA damaging drugs, and microtubule toxins that induce
the apoptosis of human tumors. Future studies will evaluate the ability
of EGF to protect cells against chemotherapeutic drugs in
vitro and in xenograft tumor models in nude mice.
 |
FOOTNOTES |
*
Supported by National Institutes of Health Grants DK 48845, DK 37871, GM 30324, and CA 58157.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
A Leukemia Society Fellow. To whom correspondence should be
addressed: Program in Molecular Signal Transduction, Division of Basic
Sciences, National Jewish Medical and Research Center, 1400 Jackson
St., Denver, CO 80206. Tel.: 303-398-1772; Fax: 303-398-1225; E-mail:
johnsonlab{at}njc.org.
 |
ABBREVIATIONS |
The abbreviations used are:
EGF, epidermal
growth factor;
ERK, extracellular signal-regulated kinase;
HEK, human
embryonic kidney epithelial cells;
MEK, mitogen-activated protein
kinase kinase.
 |
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