The most frequently found alteration of the
epidermal growth factor receptor (EGFR) in human tumors is a deletion
of exons 2-7. This receptor, termed EGFRvIII, can transform NIH 3T3
cells, and the frequent expression of this variant implies that it
confers a selective advantage upon tumor cells in vivo.
Although EGFRvIII is a constitutively activated tyrosine kinase, there
is no increase in Ras·GTP levels and low levels of mitogen-activated
protein kinase activity in NIH 3T3 cells expressing this variant. We
investigated whether phosphatidylinositol (PI) 3-kinase was an effector
in transformation by the EGFRvIII. High levels of PI 3-kinase activity were constitutively present in EGFRvIII-transformed cells and were
dependent upon the kinase activity of the receptor. While mitogen-activated protein kinase activity was quickly down-regulated to
basal levels after 12 h of continuous EGFR activation, there was a
3-fold increase in PI 3-kinase activity in cells expressing normal EGFR
and an 8-fold increase in cells expressing EGFRvIII after 48 h.
This increased activity may reflect enhanced binding to EGFRvIII and
the presence of novel PI 3-kinase isoforms. Treatment with the PI
3-kinase inhibitors wortmannin and LY294002 blocked both
anchorage-independent growth and growth in low serum media and also
resulted in morphological reversion of EGFRvIII-transformed cells.
These results support an essential role for PI 3-kinase in
transformation by this EGFR variant.
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INTRODUCTION |
Overexpression of the
EGFR1 has been implicated in
the pathogenesis of many human tumors, including those derived from the brain, breast, lung, ovary, prostate, and skin (1, 2). A number of
alterations within the EGF receptor gene that result in aberrant
protein products have also been described, primarily in human glial
tumors (3, 4). The most common alteration of the EGF receptor gene is a
deletion encompassing exons 2-7 (3-5) (referred to as EGFRvIII,
EGFR, or de2-7EGFR) (6-8). This receptor variant has
subsequently been identified in other types of primary human brain
tumors as well as breast carcinomas, non-small cell lung carcinomas,
and ovarian tumors (6, 9). This deletion results in a receptor with a
267-amino acid deletion in the extracytoplasmic domain near the amino
terminus. The frequent expression of this variant in various tumors
types suggests a strong selective advantage conferred upon tumor cells
in vivo (7, 10).
Because the deletion occurs after the signal sequence, the EGFRvIII can
be properly targeted to the membrane, and the remaining extracellular
portion is glycosylated (11, 12). While the EGFRvIII has been detected
on the cell surface of both tumor cells in vivo (13, 14) and
a number of different transfectants (12, 14, 15), significant
accumulations in the perinuclear area have also been observed, which
suggests aberrant trafficking of this receptor variant (11, 15). A
number of other functional differences between EGFRvIII and normal EGF
receptor have been characterized. Although EGFRvIII fails to bind EGF,
the receptors can dimerize, and the tyrosine kinase in the
intracellular portion of the receptor is constitutively activated (15,
16), so that the receptor undergoes autophosphorylation as well as
phosphorylating substrates such as Shc (15-17). While EGFRvIII can
bind Grb2·mSos complexes, implicating activation of the Ras/Raf/MAP
kinase pathway (18, 19), we found no increase in Ras·GTP levels and
very low levels of MAP kinase activity (15, 20), so this is unlikely to
be the primary proliferative and transforming signal propagated by
EGFRvIII. Interestingly, there are two points in the signal transduction pathway at which MAP kinase activation is down-regulated. Overexpression of EGFRvIII leads to decreased levels of Shc and Grb2,
which could reduce Ras activation, and there is an increase in MAP
kinase phosphatase activity in these cells as well (15, 20).
The normal EGF receptor is capable of initiating a variety of signaling
cascades upon ligand activation. One such effector whose importance in
tumorigenesis is becoming increasingly apparent is phosphatidylinositol
3-kinase (PI 3-kinase). PI 3-kinase was first shown to be important in
transformation by the observations that it associates with polyoma
virus middle T protein upon phosphorylation by c-Src, and that mutants
of middle T which fail to recruit PI 3-kinase activity are impaired in
their tumorigenic activity (22-24). In addition, PI 3-kinase
activation has been shown to be essential for induction of DNA
synthesis by EGF (25). We therefore investigated the possible role
played by this enzyme in transformation by the EGFRvIII, and we now
report that PI 3-kinase is constitutively activated in
EGFRvIII-transformed cells and is essential for transformation by this
receptor variant.
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EXPERIMENTAL PROCEDURES |
Cell Lines and Materials--
The cell lines which overexpress
the normal EGF receptor (CO12 20c2/b) or EGFRvIII (HC2 20d2/c) and the
vector-only control line LTR b2 were derived from NIH 3T3 cells and
maintained as described previously (15). Growth experiments in
monolayer and soft agar were performed as described (15). Media,
recombinant human EGF, and PDGF-BB were from Life Technologies, Inc.
Tyrphostin AG1478 was from Calbiochem, and the PI 3-kinase inhibitors
LY294002 and wortmannin were from Biomol (Plymouth Meeting, PA). Stocks (20 mM) were dissolved in Me2SO and stored at
20 °C and were diluted in Me2SO so that the same amount of
Me2SO (0.1%) was present in all conditions. Lipid substrates
and standards were from Sigma.
[
-32P]ATP, 125I-goat anti-mouse IgG, and
anti-rabbit IgG were from NEN Life Science Products. Normal human
fibroblast lysate, anti-phosphotyrosine monoclonal antibody PY20, and
anti-panERK monoclonal antibody were from Transduction Laboratories
(Lexington, KY), and the polyclonal and monoclonal antibodies to PI
3-kinase p85 were from U.B.I. (Lake Placid, NY). Anti-EGF receptor
monoclonal antibody was from Promega (Madison, WI), and anti-GST
antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Polyclonal antibodies to the EGFRvIII and Gab1 were produced and
affinity-purified as described previously (13, 26). Silica Gel 60 plastic sheets were from EM Science (Gibbstown, NJ), and all other
materials were from Fisher.
Immunoprecipitation and PI 3-Kinase
Assay--
Immunoprecipitations and PI 3-kinase assays were carried
out as described previously (26, 27) with slight modifications. Cells
were washed twice with ice-cold phosphate-buffered saline and lysed in
Nonidet P-40 lysis buffer (1% Nonidet P-40, 10% glycerol, 137 mM NaCl, 20 mM Tris·HCl, pH 7.4, 1 mM MgCl2, 1 mM sodium
orthovanadate, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 100 µg/ml
phenylmethylsulfonyl fluoride). Lysates were centrifuged at 12,000 × g for 10 min at 4 °C, and the protein concentration
was determined by Bio-Rad DC protein assay. Lysates were adjusted to 1 µg/ml with lysis buffer, and equal amounts were precleared with
nonspecific IgG bound to bovine serum albumin-blocked Protein G
Plus/Protein A-agarose (Oncogene Research, Cambridge, MA). Precleared
lysates were subjected to immunoprecipitation with the specified
antibodies prebound to Protein G Plus/Protein A-agarose by rocking at
4 °C overnight, and the immune complexes were washed three times
with lysis buffer, twice with 0.5 M LiCl in 100 mM Tris·HCl (pH 7.5) plus 100 µM sodium
orthovanadate, and twice with reaction buffer (25 mM
Tris·HCl, pH 7.5, 100 mM NaCl, 6.25 mM
MgCl2, 0.625 mM disodium EDTA). The beads were
resuspended in 40 µl of reaction buffer, and 10 µl of substrate
mixture (phosphatidylinositol and phosphatidylserine dispersed by
sonication in 10 mM HEPES, 1 mM EGTA, pH 7.5)
was added. The tubes were incubated at room temperature for 10 min and
reactions were initiated by addition of 30 µCi/tube
[
-32P]ATP in 5 µl of 500 µM ATP and
terminated by addition of 80 µl of 1 M HCl after 10 min.
Phospholipids were extracted with 160 µl of
CHCl3:CH3OH (1:1) and the CHCl3
layer saved. The lipids were desiccated, and the pellets were
redissolved in 12 µl of CHCl3:CH3OH (2:1) and
chromatographed on thin layer chromatography plates (precoated with
potassium oxalate and baked at 100 °C for 1 h just before use)
in CHCl3:CH3OH:2.5 M
NH4OH (9:7:2, v/v). Spots corresponding to PI 3-phosphate
were quantitated on a PhosphorImager 445SI (Molecular Dynamics,
Sunnyvale, CA).
GST Fusion Proteins, Far-Westerns, and Pulldowns--
A
construct containing the N- and C-SH2 domains of human PI 3-kinase
p85
was produced by reverse transcription polymerase chain reaction
using primers flanking cDNA nucleotides 1002-2214 (amino acids
321-724). The polymerase chain reaction product was cloned in frame
into the BamHI/EcoRI sites of pGEX 5x-3
(Pharmacia Biotech Inc.), and fusion protein (GST·PI3K-SH2) was
produced and purified as per Pharmacia protocols. For GST pulldowns,
lysates were precleared with GST bound to glutathione beads in Triton X-100/glycerol/HEPES buffer (15) containing 5 mM
dithiothreitol and then rocked with GST·PI3K-SH2 prebound to
glutathione beads at 4 °C overnight. The beads were then washed
three times with Triton X-100/glycerol/HEPES + 5 mM
dithiothreitol and resuspended in SDS-polyacrylamide gel
electrophoresis sample buffer. Electrophoresis and immunodetection were
as described previously (15). For far-Westerns, blots were probed with
the indicated concentrations of fusion protein for 1 h, washed
three times for 5 min each with 0.1% Tween 20 in Tris-buffered saline,
pH 7.5, incubated with anti-GST antibody at 1 µg/ml for 1 h,
washed, incubated with 0.3 µCi/ml 125I-anti-mouse IgG for
30 min, and then washed again. All incubations were in 1% bovine serum
albumin/0.1% Tween 20 in Tris-buffered saline, pH 7.5, containing 5 mM dithiothreitol at 4 °C.
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RESULTS |
PI 3-Kinase Is Constitutively Activated in EGFRvIII-transformed
Cells--
To study signaling in a cell line expressing EGFRvIII we
used the NIH 3T3 transfectant clone HC2 20d2/c, and for comparison we
used CO12 20c2/b, which overexpresses normal human EGF receptor (15).
PI 3-kinase activity was initially examined in serum-starved cells with
or without stimulation with 100 ng/ml EGF. As an estimate of the
maximal PI 3-kinase activity we also stimulated cells with 50 ng/ml
PDGF for 5 min, as PDGF caused the highest stimulation of PI 3-kinase
in NIH 3T3 cells among several growth factors or combination of growth
factors tested (data not shown). Analysis of the total
phosphotyrosine-associated PI 3-kinase activity revealed a very low
basal activity in vector-only control transfectants (LTR b2) and CO12
cells. We observed a much greater stimulation of PI 3-kinase by PDGF
than EGF, which was similar in both LTR b2 and CO12 20c2/b cell lines
(Fig. 1). In contrast, HC2 20d2/c cells
exhibited a high basal level of PI 3-kinase activity which was similar
to the maximal activity observed in PDGF-stimulated CO12 20c2/b or LTR
b2. As expected, HC2 20d2/c cells showed no response to EGF. There was
only a slight increase by PDGF, indicating that the basal activity
approached the highest obtainable in these cells (Fig. 1).
Preincubation of cells with tyrphostin AG1478, a highly specific
inhibitor of the EGF receptor kinase (28), reduced the
phosphotyrosine-associated PI 3-kinase activity in cells expressing
either receptor (Fig. 2), suggesting that
the EGF receptor tyrosine kinase activity is directly involved in PI
3-kinase activation in these cells. Wortmannin, a fungal metabolite, is
an irreversible inhibitor of PI 3-kinases, as it binds covalently to
the active site of the enzyme (29). Type I PI 3-kinases are especially
sensitive to wortmannin, with IC50 values in the low nanomolar range (21, 29). The IC50 of the PI 3-kinase
activity in phosphotyrosine immunoprecipitates from HC2 20d2/c cells
for wortmannin was less than 10 nM (Fig. 2,
right), indicating that this activity is due to a type I PI
3-kinase.

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Fig. 1.
PI 3-kinase is constitutively activated in
cells expressing EGFRvIII. NIH 3T3 transfectants overexpressing
the EGFRvIII (HC2 20d2/c), the normal human EGF receptor
(CO12 20c2/b), or vector-only controls (LTR b2)
were serum-starved for 1 day and then treated with DMEM only ( ), 100 ng/ml EGF (+E), or 50 ng/ml PDGF-BB (+P) for 5 min and lysed. Anti-pTyr immunoprecipitates were analyzed for PI
3-kinase activity as described under "Experimental Procedures."
Autoradiogram of thin layer chromatography plate exposed overnight is
shown. The large arrow indicates the position of
phosphatidylinositol 3-phosphate, while the small arrow
indicates the origin. Fold activation relative to each cell line's
respective unstimulated control was determined by PhosphorImager
quantitation of the PI 3-phosphate spot.
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Fig. 2.
The PI 3-kinase activity in cells
overexpressing normal EGF receptor or EGFRvIII is dependent upon the
kinase activity of the receptor. A, serum-starved cells were
treated with DMEM only or DMEM + 2 µM tyrphostin AG1478
(AG1478) for 3 h; CO12 20c2/b cells were then
stimulated with EGF for 5 min prior to lysis. Anti-pTyr
immunoprecipitates were assayed for PI 3-kinase activity as described
previously. The PI 3-kinase activities were normalized to that in the
absence of inhibitors for each cell line. B, PI 3-kinase
activity of replicate anti-pTyr immunoprecipitates from control HC2
lysate containing the indicated nanomolar concentrations of wortmannin
(WORT) in the in vitro PI 3-kinase
reactions.
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PI 3-Kinase Activity Is Not Down-regulated by the Constitutive
Activity of the EGFRvIII--
The fact that MAP kinase activity is
down-regulated in HC2 20d2/c cells implies that it plays little role in
transformation by EGFRvIII. If PI 3-kinase activity were important for
transformation by EGFRvIII, then it should not show evidence of long
term down-regulation. Because this receptor is constitutively active we
devised a protocol using tyrphostin AG1478 to regulate EGFRvIII.
Treatment of HC2 20d2/c cells with 2 µM AG1478 resulted
in a loss of both EGFRvIII tyrosine phosphorylation and PI 3-kinase
activity, but maximum inhibition of PI 3-kinase required serum
starvation (Fig. 3A). We found
that the daily addition of 2 µM AG1478 for 3 days
followed by 1 day of serum starvation in the presence of AG1478
resulted in confluent monolayers and a reduction in the level of PI
3-kinase activity to near that of quiescent CO12 20c2/b cells (see
below) without a reduction in EGFRvIII levels (Fig. 6F and
data not shown). The prolonged AG1478 treatment was required to ensure
down-regulation of a MAP kinase phosphatase which would otherwise
prevent MAP kinase activation in these cells (20). While Han et
al. have reported that the EGFRvIII is more sensitive to AG1478
than the wild-type EGF receptor (30), we did not observe such
differential sensitivity in the present study (Fig. 3A and
data not shown).

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Fig. 3.
PI 3-kinase activity is not down-regulated in
cells expressing EGFRvIII in contrast to MAP kinase. A,
effect of AG1478 on EGFRvIII tyrosine phosphorylation and PI 3-kinase
activity. HC2 20d2/c cells were treated with 2 µM AG1478
or Me2SO only in 10% CS for 3 days followed by 1 day in DMEM.
Lysates prepared on each day were quantitated and aliquots were
analyzed for tyrosine phosphorylation of EGFRVIII by Western blotting
and PI 3-kinase activity in anti-pTyr immunoprecipitates. The
EGFRvIII-pTyr bands and PI phosphate spots were quantitated, and the
percent inhibition relative to the respective controls was calculated
as [(control treated)/control] × 100. Open bars,
pTyr on EGFRvIII; gray bars, PI 3-kinase activity.
B, prolonged AG1478 treatment of cells expressing EGFRvIII
restores the activation of MAP kinase by PDGF and reveals transient MAP
kinase activation by the EGFRvIII. Cells were grown for 3 days in 10%
CS followed by 1 day of serum starvation with daily additions of
Me2SO only (DMEM) or 2 µM tyrphostin
AG1478 in Me2SO (T4d), without ( ) or with addition
of EGF (E) or PDGF (P) for 5 min prior to lysis.
T4d-6h, cells were treated as described for T4d
but changed to DMEM only for 6 h prior to lysis. Lysates were
separated on 8.5% gels, and blots were probed with anti-panERK antibody. Arrows indicate the position of activated MAP
kinase (pp42 ERK2). C, kinetics of PI 3-kinase activation in
cells expressing EGFRvIII upon release from AG1478. HC2 20d2/c cells
were treated with AG1478 as described for T4d, then washed
once and changed to DMEM only. CO12 20c2/b cells were grown in the same
way but without tyrphostin treatment. Replicate plates were lysed at
the indicated times after tyrphostin removal (HC2) or EGF
addition (CO12) and subjected to anti-phosphotyrosine
immunoprecipitation, and PI 3-kinase activity was determined as
described under "Experimental Procedures." The PI 3-kinase activity
at each time point was normalized to the respective unstimulated
(time 0) activity of each cell line, and the means ± S.E. from three independent experiments are presented. D,
kinetics of MAP kinase activation in cells expressing EGFRvIII upon
release from AG1478. Aliquots of the same lysates described in
B were analyzed for MAP kinase activation as described in
B. The activated (pp42) and inactive (p42) ERK2 bands were quantitated with the PhosphorImager, and the percent MAP kinase activation was calculated ([pp42/(pp42+p42)] × 100%).
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We confirmed the specificity of AG1478 for EGF receptor signaling.
Treatment of serum-starved CO12 20c2/b cells with either EGF or PDGF
resulted in a robust activation of MAP kinase, whereas cells pretreated
with AG1478 showed MAP kinase activation only in response to PDGF (Fig.
3B). While neither EGF nor PDGF resulted in MAP kinase
activation in serum-starved HC2 20d2/c cells, AG1478-treated HC2 20d2/c
exhibited strong MAP kinase activation in response to PDGF (Fig.
3B). Removal of the drug after serum starvation resulted in
activation of MAP kinase in HC2 20d2/c concurrent with a recovery of
the tyrosine kinase activity of EGFRvIII (Fig. 3B).
Tyrphostin removal alone caused no MAP kinase activation after similar
treatment of CO12 20c2/b cells, but as expected the cells did recover
the ability to respond to EGF (Fig. 3B). These results
demonstrate that EGFRvIII can signal through the MAP kinase pathway and
that AG1478 does not interfere with other events that are
necessary for MAP kinase activation.
We then evaluated the kinetics of PI 3-kinase and MAP kinase activation
in these transfectants. EGF stimulation of quiescent CO12 20c2/b
resulted in a very rapid activation of PI 3-kinase with the maximum
occurring between 5 and 30 min. This was followed by a decline in
activity by 12 h to ~3-fold above basal, which remained at this
level for at least 48 h (Fig. 3C). MAP kinase activation also peaked between 5 and 30 min but declined to basal activity after 12 h (Fig. 3D). In contrast, when
AG1478-treated HC2 20d2/c cells were released from tyrphostin there was
a large increase in PI 3-kinase activity, which rose to ~8-fold above basal and persisted for at least 48 h (Fig. 3C). There
was only a modest activation of MAP kinase which returned to nearly
basal activity within 6 h (Fig. 3D). Taken together,
these results indicate that cells expressing EGFRvIII can tolerate a
prolonged high level of PI 3-kinase activity, suggesting a role for
this enzyme in long term processes. On the other hand, the rapid
down-regulation of MAP kinase activity in HC2 20d2/c cells following
activation of EGFRvIII further suggests that it has no long term role
in neoplastic transformation.
PI 3-Kinase is Constitutively Associated with the EGFRvIII and
Gab1--
We wished to explore how EGFRvIII effected PI 3-kinase
activation. PI 3-kinase is activated by the binding of the two SH2 domains of the p85 subunit to pYXXM motifs (21, 27). The
five major autophosphorylation sites on the EGF receptor do not fit these motifs, so the direct association of PI 3-kinase activity is
usually low (27, 31). Under certain circumstances, Src can
phosphorylate the EGF receptor on Tyr920, which has the
consensus pYXXM motif for PI 3-kinase binding (31). The EGF
receptor can also phosphorylate the Gab1 docking protein, which in turn
can recruit PI 3-kinase activity (26). Anti-EGF receptor
immunoprecipitates from EGF-stimulated CO12 20c2/b cells showed a
~3-fold increase in the associated PI 3-kinase activity with a
similar increase in anti-Gab1 immunoprecipitates. Overall, the activity
associated with total anti-phosphotyrosine immunoprecipitates increased
~8-fold (Fig. 4, A and
C). PDGF did not increase the PI 3-kinase activity
associated with the EGF receptor and resulted in only a small increase
in the activity in Gab1 immunoprecipitates while stimulating the total
phosphotyrosine-associated PI 3-kinase activity ~60-fold (Fig.
4C). In contrast, unstimulated HC2 20d2/c cells had very
high levels of PI 3-kinase activity associated with the EGFRvIII and
Gab1, and EGF caused no significant change in PI 3-kinase activity in
these cells. PDGF addition resulted in only a ~2-fold increase in the
total phosphotyrosine-associated activity and actually caused a
decrease in the EGFRvIII-associated activity (Fig. 4, A and
B). These results indicate that the binding of PI 3-kinase
to EGFRvIII, like the normal EGFR, is of lower affinity than the
binding to the PDGF receptor, as PI 3-kinase could be recruited away
from the EGFRvIII by the two high affinity p85 binding sites on the
activated PDGF receptor.

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Fig. 4.
PI 3-kinase activity associates with the
EGFRvIII and Gab1. A, serum-starved CO12 20c2/b and HC2
20d2/c cells were lysed without stimulation ( ) or after 5 min of
stimulation with EGF (+E) or PDGF (+P).
Anti-phosphotyrosine ( pTyr), anti-EGF receptor
( EGFR), or anti-Gab1 ( Gab1)
immunoprecipitates were analyzed for PI 3-kinase activity as described.
Arrows indicate the position of PI 3-phosphate.
B, effect of EGF and PDGF on the PI 3-kinase activity in HC2
20d2/c cells associated with phosphotyrosine, the EGFRvIII, and Gab1
relative to the respective unstimulated controls. C, effect
of EGF and PDGF on the PI 3-kinase activity in CO12 20c2/b cells
associated with phosphotyrosine, the normal EGF receptor, and Gab1
relative to unstimulated controls. Values in B and
C are the means ± S.E. of three independent
experiments. The absolute amount of phosphotyrosine-associated PI
3-kinase activity in serum-starved HC2 20d2/c cells was approximately
the same as that in PDGF-treated CO12 20c2/b cells.
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Direct Association of PI 3-Kinase p85 Is Constitutive and
Phosphotyrosine-dependent--
To determine whether the PI
3-kinase activity associated with the EGFRvIII could be accounted for
by a direct interaction with p85, we performed precipitations using
GST-PI3K-SH2. The resulting blots were probed with anti-phosphotyrosine
antibody, confirming that there was an association that was dependent
upon tyrosine phosphorylation of the normal and mutant receptors (Fig. 5A). Western blotting with
anti-EGF receptor antibodies confirmed the identity of the normal and
mutant EGF receptor as the major GST-PI3K-SH2-associated proteins in
these experiments (data not shown). Far-Westerns were also performed
using GST-PI3K-SH2 fusion protein, also confirming that association
with the normal EGF receptor and the PDGF receptor was growth
factor-dependent, but the association with the EGFRvIII was
independent of EGF.

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Fig. 5.
Direct association of the p85 subunit of PI
3-kinase with normal EGF receptor and EGFRvIII. A,
association of PI 3-kinase p85 with the EGF receptor is dependent upon
tyrosine phosphorylation. Lysates from serum-starved ( ), EGF
(+E) or PDGF (+P) stimulated cells were used in
pulldowns with GST-PI 3-K-SH2 fusion protein as described under
"Experimental Procedures," and the blot was probed with
anti-phosphotyrosine ( pTyr). The upper arrow
indicates the position of the normal EGF receptor and the PDGF receptor which are not resolved on this blot, and the lower arrow
indicates the EGFRvIII. B and C, overexpression
of EGFRvIII induces elevated expression of molecules immunologically
related to p85. B, normal human fibroblast (NHF),
CO12 20c2/b (CO12), and HC2 20d2/c (HC2) lysates
were separated by SDS-polyacrylamide gel electrophoresis, and the blot
was probed with monoclonal antibody (U.B.I. 05-212) specific for the
N-SH3 domain of p85 . This monoclonal antibody recognizes the same
p85 band in all three cell lines (arrow). C, the
same blot was re-probed with a polyclonal antibody to rat p85 (U.B.I.
06-497). This antibody detected 55- and 50-kDa bands (small
arrows) as well as p85. Numbers on the left of all
blots indicate positions of molecular mass markers (kDa).
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Increased Expression of p85-Related Molecules in Cells
Overexpressing the EGFRvIII--
Because several isoforms of PI 3-K
adapters, including p85
and p85
, have been described, we wondered
which form was expressed in these cells. Western blotting of CO12
20c2/b and HC2 20d2/c lysates with an antibody specific for the N-SH3
domain of p85
detected the same band of ~85 kDa in all three lines
and revealed increased expression of this isoform in HC2 20d2/c cells
(1.64 ± 0.35-fold relative to CO12 20c2/b) (Fig. 5B).
A polyclonal antibody to rat p85 confirmed overexpression of p85 in HC2
20d2/c and also demonstrated elevated expression of 55 and 50 kDa bands
(Fig. 5C). These data suggest that HC2 20d2/c cells express
adapter molecules related to p85 which are involved in the activation of PI 3-kinase. These may represent recently reported alternative splice forms of p85 (32) or potentially homologous adapter subunits (33, 34).
PI 3-Kinase Activity Contributes to Growth Stimulation and
Transformation by the EGFRvIII--
To determine whether the
constitutive PI 3-kinase activity in HC2 20d2/c cells is relevant to
transformation by the EGFRvIII, the effects of PI 3-kinase inhibitors
on cell growth and morphology were examined. Addition of wortmannin to
cells grown in 1% CS resulted in a dose-dependent
inhibition of growth HC2 20d2/c cells. A 1 µM dose
resulted in about 50% inhibition of HC2 20d2/c cells and nearly
completely abolished the growth stimulation of EGF of CO12 20c2/b cells
(Fig. 6A). Similar results
were also obtained with the PI 3-kinase inhibitor LY294002 (Fig.
6B). The extent of growth inhibition correlated with the
degree of inhibition of PI 3-kinase activity by both drugs (Fig.
6C). The PI 3-kinase inhibitors also caused partial
morphological reversion of HC2 20d2/c cells (compare Fig. 6,
D and E) and abolished EGF-induced changes in the
morphology of CO12 20c2/b cells (data not shown). Treatment of cells
with AG1478 was more effective than the PI 3-kinase inhibitors in both
reducing PI 3-kinase activity and causing morphological reversion
(Figs. 3A and 6F, respectively). These results
indicate that PI 3-kinase activity contributes to both the growth and
morphological transformation induced by EGFRvIII.

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Fig. 6.
Growth inhibition and morphological reversion
of cells expressing EGFRvIII by PI 3-kinase inhibitors. Shown is
growth inhibition by wortmannin (A) or LY294002
(B) in monolayer culture. Cells were plated in 10% CS at
100,000/35-mm well, and the next day the media were changed to 1%
CS ± the indicated concentrations of wortmannin or LY294002.
Fresh wortmannin was added three times daily, and LY294002 was added
once daily, and the cells were trypsinized and counted with a Coulter
counter on day 3. Open bars, HC2 20d2/c; gray
bars, CO12 20c2/b. C, PI 3-kinase activity in HC2
20d2/c cells correlates with growth inhibition. Cells were treated as above, and PI 3-kinase activity in anti-pTyr immunoprecipitates of day
1 and day 3 lysates was determined. The PI 3-kinase activity relative
to the 1% CS + Me2SO control (open bars) is shown
for cells treated with 100 nM (light gray) or 1 µM (dark gray) wortmannin or with 10 µM LY294002 (black bars). D,
morphology of HC2 20d2/c in the absence and presence (E) of
5 µM LY294002 or 2 µM AG1478 (F). Cells were grown as described in B, and
phase-contrast photomicrographs of live cells are shown (× 200).
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Because anchorage-independence is the in vitro
characteristic which best correlates with tumorigenicity, we tested the
effects of the PI 3-kinase inhibitors on the growth of HC2 20d2/c in
soft agar. Wortmannin caused a dose-dependent inhibition of
colony development by HC2 20d2/c cells, which caused a ~40%
reduction in the number of colonies at a 1 µM dose, with
significant reductions in colony size at concentrations as low as 200 nM (Fig. 7A).
LY294002 was even more effective, resulting in complete abolition of
colony formation at 5 µM (Fig. 7B). Both PI
3-kinase inhibitors were even more effective in blocking
EGF-dependent soft agar colony development by CO12 20c2/b
cells than HC2 20d2/c (Fig. 7, A and B). The
smaller effect of these inhibitors on HC2 20d2/c cells may reflect the
higher endogenous level of PI 3-kinase activation in these cells. These
results indicate that PI 3-kinase activity is essential for
anchorage-independent growth in EGFRvIII-transformed cells.

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Fig. 7.
Inhibition of anchorage-independent growth of
cells overexpressing normal EGF receptor or EGFRvIII by PI 3-kinase
inhibitors. Cells were plated on 0.6% agarose underlays at
5000/35-mm dish in 0.3% agarose containing 10% CS without
(HC2) or with 20 ng/ml EGF (CO12). Wortmannin was
added daily, and LY294002 was added every 3 days. Cells were fed with 1 ml of fresh medium on days 7 and 14, and colonies were counted on day
21. A, dose response of HC2 20d2/c (open bars)
and CO12 20 c2/b (gray bars) to wortmannin in soft agar
suspension culture. B, dose response to LY294002 in soft
agar suspension culture. Asterisks above bars indicate substantial reduction in colony sizes.
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DISCUSSION |
Work in this and other laboratories has demonstrated that
expression of the EGFRvIII results in neoplastic transformation and
enhanced tumorigenicity, which is due to its constitutive kinase
activity (7, 12, 15). While activation of the normal EGF receptor
results in activation of MAP kinase via Ras (15, 18), our studies on
EGFRvIII showed only a low level of activation of the Ras-MAP kinase
pathway, which was due to decreases in Shc and Grb2 levels and
induction of a MAP kinase phosphatase (15, 20). The down-regulation of
the MAP kinase pathway has also been observed in NIH 3T3 cells
transformed by viral oncogenes such as v-src and
v-ras, and the evidence supports a role for a MAP kinase
phosphatase in these cells as well (35).
Because PI 3-kinase is essential for DNA synthesis induced by EGF (25),
we studied this enzyme in cells expressing EGFRvIII for its possible
contribution to transformation. We found that EGFRvIII-transformed
cells exhibited a high constitutive level of PI 3-kinase activity not
shown by cells overexpressing normal EGF receptor. Analysis of the
kinetics of PI 3-kinase and MAP kinase activation revealed an important
difference in the regulation of these pathways by the EGFRvIII relative
to the normal EGF receptor. While the EGF-stimulated PI 3-kinase
activity in CO12 20c2/b cells peaked quickly and declined to a moderate
level by 12 h, the activity in HC2 20d2/c cells rose more slowly
and did not decline throughout the period tested (Fig. 3C).
The slower rate of increase in PI 3-kinase activity in the
EGFRvIII-expressing cells was most likely due to the gradual decline in
tyrphostin activity, but the lack of subsequent down-regulation of PI
3-kinase activity cannot be so explained, because HC2 20d2/c cells
which have never been exposed to the drug exhibit high PI 3-kinase
activity (Fig. 1). One possibility is that cells achieve
down-regulation of EGF receptor-initiated PI 3-kinase activity
primarily by down-regulation of the number of receptors. The EGFRvIII
does not bind ligand and is not actively down-regulated despite its
constitutive activation (15, 16), and this may account for the lack of
decrease in PI 3-kinase activity in HC2 20d2/c cells. In contrast, MAP
kinase activity declines to barely above basal levels within about
12 h in both cell lines, indicating that regulation of this
pathway primarily occurs downstream of the receptor. It further
suggests that while prolonged, high level PI 3-kinase activation is
compatible with continuous growth, the prolonged, high level activation
of MAP kinase is not essential. Thus, our results are in agreement with
the recent report that prolonged MAP kinase activation in NIH 3T3 cells
results in growth arrest (36).
While we found that p85 can associate with both normal EGF receptor and
EGFRvIII, it is not clear that p85 can account for all PI 3-kinase
activity associated with EGFRvIII. Although immunoprecipitation with
anti-p85
/
antibody reduced the PI 3-kinase activity in anti-pTyr
immunoprecipitates from CO12 20c2/b cells, it did not reduce the
pTyr-associated activity in HC2 20d2/c cells (data not shown).
Furthermore, we found elevated levels of p85
in HC2 20d2/c, as well
as bands with molecular masses of 50 and 55 kDa which cross-reacted
strongly with antibody to rat p85. This suggests that the constitutive
activity of the EGFRvIII may influence the expression of PI 3-K adapter
subunits. At least five forms of regulatory subunits have been cloned
(33), including 50- and 55-kDa splice variants of p85
. Further
studies are necessary to determine whether these molecules are involved
in the PI 3-kinase activity detected in EGFRvIII transfectants.
There are several mechanisms by which PI 3-kinase may contribute to
tumorigenesis. Inhibition of PI 3-kinase activation has been shown to
block the EGF-dependent transformation of murine JB6
P+ cells (37). We found that PI 3-kinase inhibitors
inhibited both monolayer growth in low serum and anchorage-independent
growth of cells expressing normal EGF receptor and EGFRvIII. These
inhibitors also caused a partial reversion of the transformed
morphology of HC2 20d2/c and blocked the EGF-induced transformed
morphology of CO12 20c2/b. PI 3-kinase activity can influence cell
morphology, as it has been shown to affect cytoskeletal organization
(21, 22, 33). For instance, PI 3-kinase interacts with Rac·GTP, a
member of the Rho family of small G proteins which regulate the actin
cytoskeleton (38). Nagane et al. recently reported that
EGFRvIII expression reduces apoptosis of glioblastoma cells both
in vitro and in vivo (10). As activation of the
Raf/MAP kinase pathway by Ras in the absence of PI 3-kinase activity
was recently shown to promote apoptosis in fibroblasts (39), and PI
3-kinase activity has been shown to be essential for survival of a
number of cell types (40, 41), these data suggest another mechanism by
which consitutive PI 3-kinase activity contributes to tumorigenesis.
Thus, because PI 3-kinase can play a central role in growth,
morphological transformation, and the inhibition of cell death by the
both normal EGF receptor and EGFRvIII, it seems likely that enhancement
of this activity provides an important selective advantage for
EGFRvIII-expressing tumor cells in vivo.