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Tumor Biology |
Departments of Medicine [A. E. G. L., D. B., F. M. Y., C. L. A.] and Cell Biology [C. L. A.], Vanderbilt University School of Medicine, Department of Veteran Affairs Medical Center [C. L. A.], and Vanderbilt Ingram Cancer Center [C. L. A.], Nashville, Tennessee 37232, and Gilead Sciences, Foster City, California 94404 [W. M. F.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The activation of ErbB receptors results in the activation of a large network of independent signaling pathways that mediate enhanced proliferation and/or survival (2 , 3) . Overexpression of these signal transduction programs coupled with mutations in cell cycle regulators in tumor cells results in the subversion of cell cycle checkpoints and dysregulated cell proliferation (11 , 12) . In nontransformed mammalian cells, regulation of the cycle machinery is strictly controlled by several Cdks. The activity of these molecules is regulated by several mechanisms: (a) phosphorylation and dephosphorylation (reviewed in Ref. 13 ); (b) the association with specific regulatory cyclins (14) ; and (c) by the interaction with specific Cdk inhibitors (13 , 15) . On the basis of their structure and cellular targets, Cdk inhibitors can be assigned to two families: (a) the INK4 proteins that inhibit Cdk4 and Cdk6 (INK4a–d); and (b) the more broadly acting Cip/Kip family, which inhibits the catalytic subunits of the cyclin A-, D- and E-dependent kinases. The Cip/Kip family includes p21Cip1, p27Kip1, and p57Kip2 (13 , 15) . Progression through the G1-S transition is mainly regulated through the sequential activation of Cdk4, Cdk6, and Cdk2. These kinases are activated by D-type cyclins in mid-to-late G1 and by cyclin E in late G1 (13) . Cip/Kip inhibitors bind all Cdks and may prevent their activation by Cdk-activating kinases or directly inhibit their kinase activity per se. One of the Cip/Kip inhibitory molecules is p27, which was originally discovered as a Cdk inhibitory activity induced by extracellular antimitogenic signals (16 , 17) . Although p27 can interact with and inhibit recombinant cyclin D/Cdk complexes in vitro, it seems to be most effective in antagonizing the activity of cyclin E/Cdk2 complexes in vivo. By contrast, p27 seems to be required for the assembly and function of cyclin D/Cdk4 complexes in intact cells (18) .
In this study, we present evidence that ErbB2 reversibly reduces p27 and increases cyclin D1 levels through both MAPK and PI3K/Akt signaling, with neither of these pathways being dispensable for cell cycle progression. In turn, ErbB2 blockade eliminates the coordinated MAPK and PI3K/Akt inputs on p27 and cyclin D1, resulting in stabilization of p27, reduction in cyclin D1 and Cdk4, loss of Cdk2 function, and cell cycle arrest.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tubulin (Amersham
Pharmacia, Piscataway, NJ). Protein in cell lysates was measured by
bicinchoninic acid (Pierce, Rockford, IL) or Bio-Rad (Hercules, CA)
assays.
Flow Cytometric Analysis.
Proliferating BT-474 cells in IMEM/10% FCS were treated with AG1478.
Cells were harvested by trypsinization, and their nuclei were labeled
with propidium iodide as described previously (22)
. Cells
were filtered through a 95-µm pore size nylon mesh (Small Parts,
Inc., Miami Lakes, FL), and a total of 15,000 nuclei were analyzed in a
FACS/Calibur Flow Cytometer (Becton Dickinson, Mansfield, MA). DNA
histograms were modeled off-line using Modfit-LT-Software (Verity,
Topsham, ME).
Immunoblot Analysis and Immunoprecipitation.
Subconfluent monolayers (
50%) were treated for 24 h with
AG1478, LY294002, or U0126. Cells were washed twice with ice-cold PBS,
scraped in EBC lysis buffer [50 mM Tris-HCl (pH 8.0), 120
mM NaCl, 0.5% NP40, 100 mM NaF, 200
µM Na3VO4,
and 10 µg/ml each aprotinin, leupeptin, PMSF, and pepstatin], and
incubated for 20 min at 4°C while rocking. Lysates were cleared by
centrifugation (10 min at 12,000 rpm, 4°C). For immunoblot analysis,
75 µg of total protein were resolved by SDS-PAGE and transferred to
nitrocellulose membranes. Membranes were blocked with TBS-T [25
mM Tris-HCl, 150 mM NaCl (pH 7.5), and 0.05%
Tween 20] containing 5% nonfat milk and incubated overnight at 4°C
with primary antibody in TBS-T/1% nonfat milk. Blots were washed in
TBS-T and incubated with the appropriate horseradish peroxidase-linked
IgG (Amersham Pharmacia), and immunoreactive bands were detected by
chemiluminescence (Roche Molecular Biochemicals, Indianapolis, IN). For
immunoprecipitations, 1 mg (from whole cell lysates) or 500 µg (from
cytosolic and nuclear fractions) of total protein were incubated
overnight with primary antibody at 4°C; protein A-Sepharose (Sigma
Chemical Co.; 1:1 slush in PBS) was then added for 2 h at 4°C
while rocking. The precipitates were washed four times with ice-cold
PBS, resuspended in 6x Laemmli sample buffer, and resolved by SDS-PAGE
followed by immunoblot analysis.
Studies with Antisense Oligonucleotides and Cyclin D1 Adenovirus.
The sequences of the p27 antisense and the mismatch control
oligonucleotides were TGG CTC TCX TGC GCC (GS5413) and TGG CTC XCT TGC
GCC (GS5585), respectively (23)
. The oligonucleotides
(final concentration, 30 nM) were heated for 5 min at
65°C in serum-free medium to denature their secondary structure, then
mixed with 2 µg/ml cytofectin GS3815 (Gilead Sciences) in 400 ml of
serum-free minimal Eagle’s medium (MEM; Life Technologies, Inc.) in
polystyrene tubes, and incubated at room temperature for 10 min.
Thereafter, 1.6 ml of IMEM/10% FCS were added, and the resulting 2 ml
of "transfection volume" were placed on each 60-mm tissue culture
dish with BT-474 cells. Control cells were incubated under identical
conditions with 400 ml of MEM (containing 2 µg/ml cytofectin) + 1.6 ml IMEM. After 5 h, 2 ml of IMEM/10% FCS were added to
each dish, and the cells were incubated in the absence or presence of
AG1478 for 20 additional h. Cells were finally harvested and subjected
to Rb and p27 immunoblot procedures and flow cytometric analysis.
To induce expression of cyclin D1, we used an adenoviral vector encoding the human cyclin D1 gene (Ad-D1; provided by T. Meeker, University of Kentucky, Lexington, KY). Cyclin D1 gene expression is achieved by coinfection with a helper virus that is inhibited by tetracycline (Ad-tTA; Ref. 24 ). BT-474 cells (106 ) in 100 µl of Optimem (Life Technologies, Inc.)/2% FCS were incubated for 2 h at 37°C with both Ad-D1 and Ad-tTA, each at an multiplicity of infection of 200:1. Cells were then plated in IMEM/10% FCS ± 10 nM tet. In the absence of tet, high cyclin D1 expression was evident 48 h later. AG1478 was added for the second 24-h period, after which cells were harvested and prepared for immunoblot or cell cycle analyses as described above.
Cell Fractionation.
After washes and scrapping in PBS, cells were pelleted by
centrifugation (5 min, 12,000 rpm, 4°C) and incubated in a hypotonic
buffer [10 mM HEPES (pH 7.2), 10 mM KCl, 1.5
mM MgCl2, 0.1 mM EGTA, 20
mM NaF, 100 µM
Na3VO4, and 10 µg/ml of
each aprotinin, pepstatin, PMSF, and leupeptin] for 30 min at 4°C
while rocking. Cells were broken using a Dounce homogenizer (30
strokes), after which nuclei were pelleted by centrifugation (10 min,
3500 rpm, 4°C). The nuclei-free supernatant was subjected to a second
100,00 x g centrifugation for 45 min at
4°C to separate membrane (pellet) from cytosolic (supernatant)
fractions. Nuclear pellets (above) were resuspended in nuclear lysis
buffer [10 mM Tris-HCl (pH 7.5), 150
mM NaCl, 5 mM EDTA, and 1%
Triton X-100] and incubated for 1 min in a sonicating water bath,
followed by a 30-min incubation at 4°C while rocking. Twenty µg of
total cytosolic and nuclear protein were analyzed by immunoblot.
Endogenous p27 in Vitro Degradation Assay.
BT-474 cells were scrapped in p27 degradation buffer [20
mM Tris-HCl (pH 7.6), 2 mM DTT, 0.25
mM EDTA, and 10 µg/ml leupeptin and pepstatin]. Cell
suspensions were freeze-thawed three times in liquid
N2 and cleared by centrifugation (10 min, 12,000
rpm, 4°C). Twenty µg of total protein (supernatant) were incubated
in a total volume of 30 µl of p27 degradation buffer containing 5
mM MgCl2, 10 mM
creatine-PO4, 10 µg/ml creatine kinase (both
Roche Molecular Biochemicals), and 0.5 mM ATP (final
concentration). Samples were incubated at 30°C for 0.5–20 h, and the
degradation reaction was terminated by adding 6x Laemmli sample
buffer. The level of p27 in the samples was determined by immunoblot.
Densitometric analysis of the immunoreactive bands was carried out
using NIH Image Software (Research Services Branch, NIH, Bethesda,
MD).
Recombinant Protein Purification.
Expression and purification of His-tagged recombinant wild-type and
mutant (Thr-187 replaced by Ala) p27 (provided by M. Pagano, New York
University, New York, NY) was performed as described previously
(25)
. Briefly, bacteria containing the p27 plasmids were
grown overnight at 37°C (250 rpm) in Luria Broth medium and induced
with isopropyl-1-thio-ß-D-galactopyranoside (Fisher
Biotech, Fair, NJ) for 4 h. Bacterial pellets were resuspended in
lysis buffer [300 mM NaCl, 50 mM
Na2PO4 (pH 8.0), 1% Tween
20, and 10 µg/ml of each aprotinin, leupeptin, PMSF, soybean trypsin
inhibitor, N-tosyl-L-phenylalanine
chloromethyl ketone,
N-p-tosyl-L-lysine
chloromethyl ketone (Sigma Chemical Co.), NaF, and
Na3VO4] and lysed by
sonication. Lysates were cleared by centrifugation (20 min, 10,000 rpm,
4°C), and the supernatants were incubated with Ni-NTA Agarose
(Qiagen, Inc., Valencia, CA). Recombinant proteins were eluted in a
stepwise fashion using wash buffer [300 mM NaCl,
50 mM
Na2PO4 (pH 6.0), 10%
glycerol, 1% Tween 20, and protease/phosphatase inhibitors]
containing 100, 250, 500, or 1000 mM imidazole.
The fractions containing recombinant proteins were pooled, dialyzed
overnight against PBS, and stored at -80°C. GST-cyclin D1 fusion
protein (GST fused to the last 41 COOH-terminal amino acids of cyclin
D1; a gift from C. J. Sherr, St. Jude Children’s Hospital,
Memphis, TN) was bacterially expressed and purified as described by
Diehl et al. (26)
. Bacteria were grown
overnight in Luria Broth medium (37°C, 250 rpm), induced the next day
for 4 h with 1 mM
isopropyl-1-thio-ß-D-galactopyranoside, and
collected by centrifugation. Bacterial pellets were sonicated in lysis
buffer [20 mM Tris-HCl (pH 7.5), 150
mM NaCl, 0.5% NP40, and 1
mM PMSF], cleared by centrifugation (20 min,
10,000 rpm, 4°C), and incubated with glutathione-coupled-agarose (1:1
slush in PBS; Sigma Chemical Co.) for 1 h at 4°C. GST-cyclin D1
fusion proteins were eluted from the glutathione-agarose beads using
GSK-3ß kinase buffer (below) containing 4 mM
reduced glutathione (Sigma Chemical Co.).
In Vitro Kinase Assays.
Cells were washed with ice-cold PBS and lysed in EBC buffer. One mg of
total protein was precipitated overnight at 4°C with MAPK, Cdk2, or
GSK-3ß antibodies and protein A-Sepharose. Immunoprecipitates were
washed four times with ice-cold PBS, two times with kinase buffer
[Cdk2 kinase buffer: 50 mM HEPES (pH 7.5), 10
mM MgCl2, 2.5 mM EGTA, 1
mM DTT, 0.1 mM NaF, 0.1 mM
Na3VO4, and 1
mM ATP; MAPK kinase buffer: 30 mM HEPES (pH
7.4), 15 mM MgCl2, and 1
mM ATP; GSK-3ß kinase buffer: 50 mM HEPES (pH
7.5), 10 mM MgCl2, 1 mM
EGTA, 1 mM DTT, 1 mM PMSF, 0.4 mM
NaF, 0.4 mM
Na3VO4, and 1
mM ATP], and finally resuspended in 20 µl of its
respective kinase buffer. Cdk2 precipitates were assayed for their
kinase activity by adding 1 µg of HH1 (Roche Molecular Biochemicals),
and MAPK activity, by adding 5 µg of MBP (Upstate Biotechnology).
Cdk2 or MAPK precipitates were also incubated with 10 µg of
His-tagged wild-type or T187A p27 proteins. GSK-3ß kinase activity
was determined against 5 µg of recombinant GST-cyclin D1. Kinase
reactions were performed in a final volume of 30 µl in the presence
of 5 µCi of [
-32P]ATP (specific activity,
3000 Ci/mmol; Amersham Pharmacia) for 45 min at 30°C, 25 min at
25°C, or 30 min at 30°C for Cdk2, MAPK, and GSK-3ß, respectively.
Cyclin D1/Luciferase Reporter Assays.
BT-474 cells in IMEM/10% FCS were seeded in 24-well plates
(2 x 105 cells/well). Twenty-four
h later, the cells were transfected with 250 ng of pGL3 vector
containing the cyclin D1 promoter (a gift from L. Sealy, Vanderbilt
University, Nashville, TN) and 1 ng of pRL-CMV construct
(Promega) using Fugene-6 Transfection Reagent (Roche Molecular
Biochemicals). Twenty-four h after transfection, 10 µM
AG1478 or 5 µM U0126 were added to the cells. After a
24-h incubation with these inhibitors, the cells were washed with PBS,
lysed, and analyzed for firefly Luc and Renilla reniformis
Luc activities using the Dual Luciferase Reporter Assay system
(Promega) according to the manufacturer’s instructions in a Monolight
2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA).
Luc activity was normalized to Renilla reniformis Luc and
expressed as fold-induction above control.
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RESULTS |
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. Treatment with the protein synthesis inhibitor cycloheximide revealed
a half-life for p27 of approximately 12 and 4 h in clones 11 and
18, respectively, correlating inversely with the levels of
phosphorylated ErbB2. Levels of the Cdk inhibitor were minimally
reduced at 24 h in MCF-7/neo cells treated with cycloheximide
(Fig. 1)
, suggesting that overexpression of ErbB2 increased the
turnover of p27. The steady-state levels of cyclin D1 protein were
similar in all three cell lines (not shown).
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. Within 3 h (in BT-474) and <8 h (in SKBR-3) of removal of
AG1478, ErbB2 was phosphorylated and cyclin D1 levels were markedly
increased. However, the cells remained in G1 for
>24 h after removal of AG1478 and reentered into S-phase at 48 h,
the time at which Rb was hyperphosphorylated and p27 levels were
down-regulated (Fig. 2)
. In SKBR-3 cells, p21 was only detectable after
removal of AG1478 (Fig. 2B)
. The content of cyclin A was not
altered in either cell line (data not shown). These results suggested
that AG1478 reversibly inhibited the ErbB2 kinase in vivo
and that p27 was probably limiting for cell cycle progression.
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. In cells treated with cytofectin alone or mismatch
oligonucleotides, 1–5 µM AG1478 induced an
increase in the G1 fraction (from 71 to 87; 84%)
and a decrease in the proportion of cells in S-phase (from 18 to 6;
11%). These did not occur in cells preincubated with antisense p27
oligonucleotides (Fig. 3B)
and in which the levels of p27
had been down-regulated. Cdk2 and Cdk4 protein levels were not altered
by either oligonucleotide (not shown). Similar results were obtained
three times, suggesting a critical role for p27 in the
G1 arrest that follows blockade of the ErbB2
kinase.
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, consistent with their reported low levels of EGF receptors as
measured by more sensitive methods in other studies (30
, 31) . On the other hand, ErbB2 was only detectable in the
MCF-7/ErbB2–18 and BT-474 cells but not in MCF-7/neo cells. The ErbB2
band comigrated with a Mr 180,000
P-Tyr band in MCF-7/ErbB2–18 and BT-474 cells; this band was
eliminated by treatment with AG1478, suggesting that ErbB2 kinase
activity was constitutive in these cell lines. The content of both
total MAPK and total Akt were similar in all three cell lines. However,
active MAPK and active Akt as measured by antibodies specific for
phospho-ERK1/2 and phospho-Ser473 Akt, respectively, were higher in
MCF-7/ErbB2–18 and BT-474 than in MCF-7/neo cells. A 24-h treatment
with 10 µM AG1478 reduced both active MAPK and
active Akt without changes in total MAPK and total Akt. To follow the
AG1478 mediated down-regulation of cyclin D1, we examined Cdk levels.
Cdk2 protein levels were similar in all three cell lines. However, Cdk4
levels were lower in the two ErbB2-overexpressing lines and were
further reduced by inhibition of ErbB2 with AG1478 (Fig. 4)
.
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was explained by modulation of
p27/Cdks stoichiometry. In proliferating BT-474 cells, p27
coprecipitated with both Cdk4 and Cdk2, with higher levels of p27
precipitating with Cdk4 than with Cdk2 antibodies (Fig. 5A)
. Upon interruption of ErbB2 signaling with AG1478, the
association of Cdk2 with p27 was markedly increased with significant
inhibition of the amount of p27 that precipitated with Cdk4 antibodies
or vice versa. Cdk2 protein levels were not altered, whereas
Cdk4 levels were markedly reduced by AG1478, accounting in part for the
diminished interaction with p27 (Fig. 5A)
. Because p27 can
either inhibit cyclin E/Cdk2 activity or serve as a substrate for
Cdk2-mediated phosphorylation, we performed Cdk2 in vitro
assays using HH1 and recombinant p27 as kinase substrates. Fig. 5B
shows that inhibition of ErbB2 reduced basal Cdk2
activity against HH1 and wild-type p27 in BT-474 cells. A mutant T187A
p27 was not phosphorylated, confirming that T187 is a Cdk2
phosphorylation site, as reported by Sheaff et al.
(32)
.
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. Kawada et al. (33)
showed that MAPK transfected into fibroblasts is capable of directly
phosphorylating p27. Therefore, we precipitated MAPK from control and
AG1478-treated cells and tested the immune complexes in kinase assays
using MBP and p27 as substrates. Fig. 5B
shows that MAPK
from proliferating BT-474 cells phosphorylates MBP and p27 directly,
whereas its activity is markedly reduced in cells arrested with AG1478.
MAPK from proliferating cells was unable to phosphorylate T187A p27,
suggesting that MAPK can also phosphorylate T187 directly. Cdk2 and
cdc2 were not detectable in MAPK precipitates used to phosphorylate p27
in vitro (not shown), arguing against the presence of a
known p27 kinase present in the MAPK precipitate. Because
phosphorylation at T187 results in proteasome-mediated degradation of
p27 (32
, 34) , we examined whether the inhibition ErbB2, by
reducing Cdk2- and MAPK-mediated phosphorylation of p27, increased the
stability of endogenous p27 using a modification of the method
described by Loda et al. (25)
. In this method,
the endogenous p27 is incubated at 30°C in the presence of ATP.
Densitometric analysis of the p27 bands revealed a half-life of <3 h
versus 
6 h in proliferating versus quiescent,
AG1478-treated cells, respectively.
Inhibition of ErbB2 Increases Nuclear Levels of p27 and Its
Association with Nuclear Cdk2.
We next evaluated how interruption of ErbB2 signaling with AG1478
affects the localization of cell cycle regulators. Fig. 6A
shows that active MAPK (P-MAPK), Cdk4, and cyclin D1 reside
mainly in the cytosol of untreated proliferating cells and are
down-regulated by AG1478. In contrast, Cdk2, cyclin E, and total MAPK
protein levels remain unaltered and can be found in both cytosol and
cell nuclei. In proliferating cells, p27 is found in both compartments,
where it coprecipitates with Cdk4 and Cdk2. Inhibition of the ErbB-2
kinase with AG1478 completely redirected p27 from cytosolic Cdk4 to
nuclear Cdk2 (Fig. 6B)
, where it may inhibit Cdk2 activity
(Fig. 5B)
and mediate cell cycle arrest. Similar to Cdk2,
cyclin E also associated at higher levels with p27 upon treatment with
AG1478 (not shown).
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confirms that AG1478 inhibited signaling through both MAPK
and PI3K/Akt. However, LY294002 only affected PI3K signaling, as
indicated by the complete inhibition of Ser-473 Akt and P-GSK-3ß
without affecting P-MAPK, whereas U0126 solely eliminated only P-MAPK.
Nonetheless, both pathways seem to have some common targets. Cdk4
levels decreased upon inhibition of PI3K/Akt and MAPK, and the combined
use of LY294002 and U0126 had a more pronounced effect. Similar to
AG1478, both LY294002 and U0126 up-regulated p27 levels. The levels of
total Akt, total MAPK, total GSK-3ß, cyclin E, and Cdk2 were not
altered by any of the inhibitors.
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. We
next investigated whether inhibition of ErbB2 signaling down-regulated
cyclin D1 at a transcriptional level. BT-474 cells were transiently
transfected with a cyclin D1/luciferase promoter construct and then
treated with AG1478, LY294002, or U0126 for 24 h. Both AG1478 and
U0126 (Fig. 7B)
but not LY294002 reduced cyclin
D1/luciferase expression 50% compared with controls. The complete
elimination of cyclin D1 by AG1478 and LY294002 (Fig. 7A)
suggested that PI3K/Akt regulated cyclin D1 posttranscriptionally. To
determine whether inhibition of PI3K/Akt resulted in GSK-3ß-mediated
phosphorylation of cyclin D1, we precipitated GSK-3ß from BT-474
cells treated with different inhibitors and tested its activity
in vitro against recombinant GST-cyclin D1. In untreated
cells and cells treated with the MEK1/2 inhibitor, GSK-3ß did not
appreciably phosphorylate GST-cyclin D1 (Fig. 7C)
,
consistent with the phosphorylated (inactive) GSK-3ß protein detected
by immunoblot (Fig. 7A)
. In contrast, in cells treated with
AG1478, LY294002, or the combination of LY294002 plus U0126, GSK-3ß
was not phosphorylated (Fig. 7A)
and, therefore, active and
capable of phosphorylating cyclin D1 in vitro (Fig. 7C)
. Despite the different biochemical effects of LY294002
and U0126 when used alone, each was able to increase the
G1 fraction and reduce the S-phase fraction of
BT-474 cells (Table 1)
, suggesting that both PI3-K/Akt and MAPK coordinately regulate
ErbB2-driven tumor cell proliferation.
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. Addition of tet completely suppressed ectopic cyclin D1 expression.
Under these conditions, the addition of AG1478 inhibited Rb
phosphorylation, increased the G1-phase fraction
(75
82%), and reduced the S-phase fraction (83%). However,
AG1478 had no effect in the absence of tet, indicating that an excess
of cyclin D1 can negate the growth arrest resulting from inhibition of
ErbB2.
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shows rapid dephosphorylation (<2 h) of ErbB2, P-MAPK, P-Akt, and
P-GSK-3ß. After 6 h, detectable levels of cyclin D1 were
eliminated, whereas Cdk4 remained stable for up to 12 h of
exposure to AG1478. The increase in p27 was first detectable at 6 h, reaching a maximum at >24 h, the time at which an increase in
G1 and a reduction in S-phase were first
detectable (Fig. 2)
. Although the levels of p27 were slightly variable
in untreated BT-474 cells, no time-dependent increase in p27 protein
levels occurred in the absence of AG1478. This temporal progression is
consistent with a model in which the increase of p27 steady-state
levels occurs as a consequence of the simultaneous inactivation of
multiple ErbB2-modulated signaling inputs.
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DISCUSSION |
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105 ErbB1 (EGFR)
sites/cell (31)
, are ErbB2 dependent in that blockade of
ErbB2 with antibodies against the receptor’s ectodomain markedly
inhibits their proliferation both in vitro and in
vivo (37)
. ErbB2 is constitutively phosphorylated in
BT-474 cells, suggesting that, in these cells, the orphan receptor may
be transactivated by ligand-activated EGFR. To block ErbB2 signaling,
we used AG1478, a small molecule quinazoline inhibitor of the EGFR
(ErbB1) kinase. In intact BT-474 cells, AG1478 inhibits the ErbB2
kinase and induces a reversible cell cycle arrest, thus providing a
useful tool to study ErbB2-driven cell cycle progression. Although
AG1478 is specific for the EGFR at submicromolar concentrations,
several arguments support its use to block EGFR/ErbB2 cross-talk. It
was shown that it induced the formation of inactive, unphosphorylated
EGFR/ErbB2 heterodimers (29)
, conditions under which ErbB2
would be unable to interact with other ErbB coreceptors. We reported
recently that AG1478 can suppress tumorigenesis in
MMTV/neu + TGF- bigenic mice (22)
,
further suggesting that EGFR kinase inhibitors can inhibit tumor cell
systems, such as BT-474 cells, in which the EGFR and ErbB2
(neu) cooperate. At the concentrations used, AG1478 had no
effect against Cdk2 (38)
, MAPK (39)
, or Akt
kinase8
in vitro, implying that its
effects on signaling pathways downstream of ErbB2 were not direct but
secondary to its action at the receptor level. Several reports link signaling pathways activated by ErbB2 with regulators of cell cycle progression. Activation of Ras/MAPK results in degradation of p27 and inhibits its ability to bind Cdk2 (33 , 40) . Both constitutive activation of MAPK (41) and overexpression of the ErbB2-homologous neu gene product (42) increase cyclin D1 transcription and expression. Via heterodimerization with ErbB3, ErbB2 can activate PI3K (3) , which phosphorylates membrane phosphoinositides at the 3' position of the inositol ring. These membrane-bound lipids recruit Akt, via its the pleckstrin homology domain, to the cell membrane, where this kinase is activated by phosphorylation in Thr-308 and Ser-473 by the 3-phosphoinositide-dependent protein kinases PDK1 and PDK2 (43) . Akt can phosphorylate and negatively regulate GSK-3ß, thereby inhibiting GSK-3ß-mediated phosphorylation of cyclin D1 at Thr-286. This phosphorylation accelerates proteasome-mediated degradation of cyclin D1, thus shortening its half-life (26) . Therefore, by activating MAPK and PI3K/Akt and indirectly disabling GSK-3ß, an excess of ErbB2 signals can modulate cyclin D1 and p27 and dysregulate the G1-to-S transition. Overexpression of ectopic ErbB2 in MCF-7 cells up-regulated both MAPK and PI3K/Akt activities and increased the turnover of endogenous p27. In these and in BT-474 cells, inhibition of the ErbB2 kinase with AG1478 blocked MAPK and Akt kinases and induced proliferation arrest, suggesting that both of these pathways are involved in ErbB2-driven cell cycle progression.
Although p27 can inhibit recombinant cyclin D1/Cdk4 complexes in
vitro, it is more effective in antagonizing the activity of cyclin
E/Cdk2 (16
, 44)
. More recent data with primary mouse
embryonic fibroblasts indicate that p27 can stabilize the assembly of
cyclin D1/Cdk4 complexes and direct this heteromeric complex to the
cell nucleus (36)
. On the other hand, in proliferating
cells, cyclin D1/Cdk4 complexes can sequester p27 and limit p27 binding
to and inhibition of cyclin E/Cdk2. Consistent with these studies, p27
was low and was associated at higher stoichiometry with Cdk4 than with
Cdk2 in proliferating than in quiescent BT-474 cells. By reducing the
amount of p27 available for Cdk2 binding, this association with cyclin
D1/Cdk4 may indirectly facilitate Cdk2-mediated phosphorylation of p27
on T187 (Fig. 5B)
to trigger its degradation. Similar
results were reported recently by Lane et al.
(45)
; growth arrest of proliferating BT-474 cells with an
inhibitory ErbB2 antibody completely redirected p27 from Cdk4 to Cdk2
complexes.
MAPK from BT-474 cells phosphorylated wild-type but not T187A p27,
implying that this residue, similar to Cdk2, may also be a target for
MAPK-mediated phosphorylation. This result, the shorter turnover of p27
in both MCF-7/ErbB2 clones compared with MCF-7/neo cells (Fig. 1)
and
in untreated versus AG1478-treated BT-474 cells (Fig. 5C)
, and the increase in steady-state p27 levels in BT-474
cells treated with the MEK1/2 inhibitor U0126 (Fig. 7A)
, all
suggest that ErbB2-activated MAPK directly may contribute to the
destabilization of p27. In addition, we showed that blockade of ErbB2
function with AG1478 inhibited the ability of BT-474 cell MAPK to
phosphorylate p27 in vitro (Fig. 5B)
. This is
unlikely because of a direct effect of AG1478 on Cdk2 because its
in vitro IC50 against Cdk2 is >100
µM (38)
. Furthermore, we did not
detect Cdk2 or cdc2, kinases well known to phosphorylate p27, in the
MAPK precipitates used to phosphorylate p27 in vitro,
suggesting that our result is not attributable to a contaminating
p27-interacting kinase. This interaction of MAPK with T187 in p27 is
not unique to BT-474 cells. We reported recently that MAPK precipitated
from MMTV/neu + TGF- tumors, in which the
neu kinase is active, was able to phosphorylate wild-type
but not T187A p27. In this same study, the ability of an
MMTV/neu + TGF- tumor lysate to degrade
recombinant p27 in vitro was completely abrogated by the
addition of U0126 (22)
. Bacterially expressed p27 has been
shown to be exclusively phosphorylated on serine by GST-Erk1 in
vitro (46)
. Ser-10 was reported recently as the major
phosphorylation site of p27 in vivo (47)
. In
this last study, however, Erk1/2 was unable to phosphorylate this site,
arguing against the serine-exclusive phosphorylation of p27 by MAPK as
reported by Alessandrini et al. (46)
. In a
recent study, overexpression of HER2 in NIH-3T3 cells enhanced
ubiquitin-mediated degradation and nuclear exclusion of p27, which were
reduced by a dominant-negative Grb-2 mutant (48)
, further
supporting a direct role for MAPK in the regulation of p27.
The previous data support the notion that MAPK-mediated destabilization
of p27 contributes to ErbB2-driven cell cycle progression. However, it
has been shown in mouse fibroblasts that activation of MEK1 is not
sufficient per se to trigger the degradation of p27 unless
cyclin D1 and Cdk4 subunits are co-overexpressed at levels achieved in
cells stimulated by serum (18)
. These cyclin D1/Cdk4
complexes can then sequester untethered p27, reduce p27/Cdk2
stoichiometry and its inhibitory threshold of Cdk2, and thereby allow
entry into S-phase. In the experimental system presented, this is
supported by the high association of transduced cyclin D1 and
endogenous p27 in highly proliferative BT-474 cells (Fig. 8)
. These
data lead to the possibility that, in addition to destabilization of
p27, other ErbB2-induced signals are required for progression into
S-phase. On the other hand, one could argue that, in addition to the
up-regulation of p27, the marked G1 arrest after
blockade of ErbB2 also requires the elimination of those additional
proliferative signals.
The marked changes in cyclin D1/Cdk4 levels observed upon inhibition of
ErbB2 support a critical role for this G1 cyclin in ErbB2-mediated cell
cycle progression. Treatment of proliferating BT-474 cells with the
MEK1/2 inhibitor U0126 inhibited cyclin D1 reporter activity while
modestly reducing cyclin D1 protein levels (Fig. 7)
. On the other hand,
treatment with the PI3K inhibitor LY294002 inhibited Akt activity,
dephosphorylated GSK-3ß, markedly reduced cyclin D1 levels, and
enhanced the ability of cellular GSK-3ß to phosphorylate cyclin D1
in vitro (Fig. 7)
. LY294002 did not inhibit MAPK activity in
BT-474, suggesting that its effect on cyclin D1 was independent of an
action on MAPK. AG1478, LY294002, and U0126 also down-regulated Cdk4,
which followed the reduction of cyclin D1 (Figs. 7
and 9)
. Unassembled
Cdk4 is known to turn over more rapidly and not accumulate
(49)
. In addition, Erk (MAPK) activity has been shown to
be required for cyclin D/Cdk4 assembly (18)
. Whether the
reduction of cyclin D1 levels and/or MAPK activity can contribute to
Cdk4 unassembly and destabilization requires further investigation
outside the scope of this report. Nonetheless, the increase in p27
induced by each LY294002 and U0126 (Fig. 7A)
could be
secondary to the strong reduction in cyclin D1/Cdk4 complexes, which
then leaves p27 untethered and able to stably bind Cdk2 at high
stoichiometry (Fig. 6B)
, thus counteracting Cdk2-mediated
phosphorylation and degradation of p27 (Fig. 5, B and C)
. These results suggest that ErbB2 may regulate cellular
cyclin D1 at two molecular levels: (a) by transcriptional
regulation via active MAPK; and (b) posttranslationally, via
PI3K/Akt, by suppression of GSK-3ß-mediated phosphorylation and
subsequent degradation of cyclin D1. Reversal of these ErbB2-mediated
effects on cyclin D1 may be required for the antiproliferative effect
of AG1478 in B-474 cells. Induction of high levels of cyclin D1
prevented the cell cycle effects of the anti-ErbB small molecule (Fig. 8)
.
Both LY294002 and U0126 independently induce G1
arrest and a 70% reduction in the S-phase fraction in BT-474 cells
(Table 1)
, implying that both MAPK and PI3K are coordinately required
for the dysregulated proliferation in ErbB2-overexpressing cells. A431
squamous cancer cells are highly dependent on autoactivated ErbB1
(EGFR) homodimers (50)
. A recent report suggested that in
these cells, PI3K/Akt signaling but not MAPK was predominantly
contributing to G1-to-S progression
(38)
. These different results with BT-474 and A431 cells
suggest that different ErbB homo- or heterodimers may not rely on
identical effector mechanisms for the subversion of the
G1-to-S transition. Notably, studies in
(ErbB-null) 32D and BAF3 cells transfected with ectopic ErbB receptors,
either singly or in combination (4
, 51)
, have not
addressed yet which molecules involved in the regulation of
G1-S are modulated by different ErbB dimers.
In summary, the data presented suggest that ErbB2 modulates cellular p27 and cyclin D1 protein levels through both Ras/MAPK and PI3K/Akt signaling. Reversible interruption of ErbB2 function, using a small molecule kinase inhibitor, inhibits MAPK (Erk) and reduces MAPK-mediated transcription of cyclin D1. In addition, PI3K/Akt signaling is inhibited, which relieves GSK-3ß from Akt-mediated inhibitory phosphorylation, thus allowing GSK-3ß to phosphorylate and degrade cyclin D1. The loss of Erk activity and cyclin D1 leave Cdk4 unassembled and possibly destabilize it. These events reduce the levels of cyclin D1/Cdk4 complexes that can sequester and titrate p27 away from binding to cyclin E/Cdk2 complexes and, in turn, inhibit Cdk2-mediated phosphorylation of Rb. Moreover, the inhibition of MAPK relieves p27 from MAPK-mediated phosphorylation on T187, further contributing to p27 stability and to its ability to bind to and inhibit Cdk2 function. In concert, these biochemical responses after ErbB2 blockade retain ErbB2-dependent cells in the G1 phase of the cell cycle. The inability of ErbB2 inhibitors to elicit these responses may translate into resistance to ErbB2-targeted anticancer therapies.
|
FOOTNOTES |
|---|
1 This work was supported by NIH Grant R01 CA80195
(to C. L. A.), a Department of Veteran Affairs Clinical Investigator
Award (to C. L. A.), and Vanderbilt-Ingram Cancer Center Support
Grant CA68485. D. B. was supported by the Robert-Bosch Foundation
(Stuttgart, Germany). A. E. G. L. is the recipient of a fellowship
award from the Susan G. Komen Foundation. 
2 Present address: Biotechnology Research
Institute, National Research Council, 6100 Royalmount Avenue, Montreal,
Quebec, H4P 2R2 Canada.
3 Present address: Dr. Margarete
Fischer-Bosch-Institut für Klinische Pharmakologie, 70376
Stuttgart, Germany.
4 Present address: Sunesis Pharmaceuticals, 3696
Haven Avenue, Suite C, Redwood City, CA 94063.
5 To whom requests for reprints should be
addressed, at Division of Oncology/Vanderbilt University, 777 Preston
Research Building, Nashville, TN 37232-6307. Phone: (615) 936-3524;
Fax: (615) 936-1790; E-mail: carlos.arteaga{at}mcmail.vanderbilt.edu
6 The abbreviations used are: EGFR, epidermal
growth factor receptor; Cdk, cyclin-dependent kinase; MAPK,
mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase;
IMEM, Improved Minimal Essential Medium; PMSF, phenylmethylsulfonyl
fluoride; tet, tetracycline; GST, glutathione
S-transferase; GSK-3ß, glycogen synthase kinase-3ß;
MBP, myelin basic protein; Luc, luciferase; HH1, histone H1; MMTV,
mouse mammary tumor virus; TGF, transforming growth factor; Erk,
extracellular signal-regulated kinase; MEK, MAPK kinase.
7 L. K. Shawver, personal communication.
8 C. L. Arteaga, unpublished data.
Received 4/25/01. Accepted 7/ 5/01.
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S. L. Moulder, F. M. Yakes, S. K. Muthuswamy, R. Bianco, J. F. Simpson, and C. L. Arteaga Epidermal Growth Factor Receptor (HER1) Tyrosine Kinase Inhibitor ZD1839 (Iressa) Inhibits HER2/neu (erbB2)-overexpressing Breast Cancer Cells in Vitro and in Vivo Cancer Res., December 1, 2001; 61(24): 8887 - 8895. [Abstract] [Full Text] [PDF]
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32,000). IP,
immunoprecipitation. C, degradation of endogenous p27.
Twenty µg of total protein from AG1478 treated (24 h) or control
BT-474 cells were incubated for variable times at 30°C in a kinase
buffer containing creatine, creatine kinase, and ATP. The p27 content
of the samples was determined by immunoblot. Left of
each panel, molecular weights of immunoreactive bands.
