From the Departments of Surgery and
Biochemistry and Molecular Biology and § Walther
Oncology Center, Indiana University School of Medicine, Indianapolis,
Indiana 46202 and the ¶ Department of Cancer Medicine, Imperial
College School of Medicine, Hammersmith Hospital,
London W12 0NN, United Kingdom
Received for publication, November 30, 2000, and in revised form, December 22, 2000
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ABSTRACT |
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Estrogen receptors (ERs) mediate most of the
biological effects of estrogen in mammary and uterine epithelial cells
by binding to estrogen response elements in the promoter region of
target genes or through protein-protein interactions. Anti-estrogens such as tamoxifen inhibit the growth of ER-positive breast cancers by
reducing the expression of estrogen-regulated genes. However, anti-estrogen-resistant growth of ER-positive tumors remains a significant clinical problem. Here we show that phosphatidylinositol (PI) 3-kinase and AKT activate ER Estrogen-induced proliferation of mammary and uterine epithelial
cells is primarily mediated by estrogen receptors
(ERs),1 which belong to
steroid/thyroid hormone superfamily of transcription factors (1). ERs,
through their estrogen-independent and estrogen-dependent activation domains (AF-1 and AF-2, respectively), regulate
transcription by recruiting coactivator proteins and interacting with
the general transcriptional machinery (2). Tamoxifen, which functions
as a cell type-specific anti-estrogen, competitively binds to ER Growth factor-dependent survival of a wide variety of
cultured cells types ranging from fibroblasts to neurons is dependent on PI 3-kinase pathway (9). Growth factor-induced activation of
transmembrane receptors (IGF-1, EGF, platelet-derived growth factor,
basic fibroblast growth factor, and heregulin) results in recruitment
of PI 3-kinase to the plasma membrane (9). In the plasma membrane, PI
3-kinase promotes generation of 3'-phosphorylated phosphoinositides,
which in turn bind to AKT. AKT bound to phosphoinositides is
translocated from cytoplasm to the plasma membrane, where it is
activated through phosphorylation. Apart from growth factors, estrogen
also activates AKT by triggering the binding of ER Cell Culture--
MCF-7 cells were grown in minimal essential
medium (MEM) containing phenol red (PR), 10% fetal calf serum (FCS),
streptomycin, and penicillin. COS-1 cells were maintained in
Dulbecco's modified MEM (DMEM) including PR, 10% FCS, streptomycin,
and penicillin.
Recombinant Plasmids, DNA Transfections, and CAT
Assays--
MCF-7 and COS-1 cells were passed 24 h prior to
transfection in PR-free DMEM + 5% charcoal/dextran-treated serum (CCS)
and transfected via the calcium phosphate method. The Phosphorylation of GST-AB by AKT in Vitro--
Amino acids
1-184 of ER AKT-overexpressing Clones and Growth Analysis--
MCF-7 cells
were transfected with either empty pcDNA3 or pcDNA3 vector
encoding CA-AKT. Transfected cell populations were selected using G418
(0.6 mg/ml) and CA-AKT expression in individual G418-resistant colonies
was verified by Western analysis. MCF-7 control (pcDNA-1) and
CA-AKT (CA-AKT-4) cells were plated in MEM with PR and 10% FCS at a
density of 2 × 105 cells/60-mm plate. After 24 h, fresh medium containing either 10 Cell Cycle Analysis and Quantitation of Apoptosis--
MCF-7
control and CA-AKT cells were plated at a density of 1 × 105 cells/60-mm dish in MEM with PR + 10% FCS. After
24 h, fresh medium was added, along with
10 Western Blot Analysis and Immunoprecipitation--
Western
blotting/immunoprecipitation of cell lysates with antibodies against
cyclin D1, PTEN (Santa Cruz), Bcl-XL, BAX, BAK (Trevigen),
Bcl-2 (PharMingen), PI3K-p110, p85 (Upstate Biotechnology, Inc.), AKT
(New England Biolabs), and Orthophosphate Labeling and Immunoprecipitation of
ER Statistical Analysis--
Data were analyzed with StatView
(version 4.1). Analysis of variance was employed to determine
p values between mean measurements. A p
value < 0.05 was deemed significant. Error bars on all histograms in this text represent standard deviations between measurements from
duplicate experiments.
AKT Confers Ligand-independent Activity to ER
A substantial (4-fold) increase in estrogen-independent activity was
observed when ERE3-TK-CAT was transfected along with PI 3-kinase, the
upstream activator of AKT (Fig. 1B). PI 3-kinase-induced ERE3-TK-CAT activity was reduced to 2.5-fold by 4-HT. CA-AKT had no
effect on basal and retinoic acid-inducible expression of
AKT Increases AF-1 Activity--
To determine the activation
domains of ER PI 3-Kinase Increases Both AF-1 and AF-2 Activity--
PI
3-kinase, apart from AKT, activates other signaling pathways including
Jun N-terminal kinase, protein kinase C Ser-167 of ER
We next determined whether basal or IGF-1-induced phosphorylation of
ER
We next compared AKT-mediated activation of wild type GAL-AB and GAL-AB
mutants lacking Ser-102, Ser-104, and Ser-106 (AB-S102N,S104P,S106A), Ser-118 (AB-S118A), Ser-167 (AB-S167A), or Ser-173 and Ser-178 (AB-S173A,S178A). Only AB-S167A did not respond to AKT, suggesting that
Ser-167 is essential for activation of AF-1 by AKT (Fig. 4C). Mutation of Ser-167 to alanine also reduced
estrogen-independent and/or estrogen-dependent activity of
full-length ER Endogenous Estrogen-regulated pS2 Gene Expression Is Up-regulated
by AKT--
To study whether CA-AKT induces the expression of
endogenous estrogen-regulated genes, we generated MCF-7 cells
overexpressing CA-AKT (Fig.
5A). We measured the
expression of pS2 and c-Myc in untreated, estrogen-, 4-HT-, and
estrogen+4-HT-treated cells. The basal level of pS2 was higher in
CA-AKT-4 cells compared with pcDNA3-2 and CA-AKT-2 cells (Fig.
5B). Estrogen-inducible expression of pS2 was higher in both
CA-AKT-2 and CA-AKT-4 cells compared with pcDNA3 cells. Note that
CA-AKT-4 cells express higher level of AKT than CA-AKT-2 cells (Fig.
5A). As with the results of transient transfection assays,
constitutive and estrogen-inducible expression of pS2 was repressed by
4-HT. Interestingly, AKT overexpression did not result in changes in
c-Myc expression.
Screening of Atlas Human Cancer cDNA expression array using RNA
from pcDNA3 and CA-AKT cells revealed up-regulation of macrophage inhibitory cytokine (MIC-1) by AKT (29). This was confirmed by Northern
blotting (Fig. 5B). MIC-1 expression was observed only in
CA-AKT-4 cells, which was increased modestly by estrogen. Interestingly, 4-HT did not suppress MIC-1 expression. Taken together, these results suggest that AKT increases either constitutive or estrogen-inducible expression of specific estrogen-responsive genes.
AKT Confers Tamoxifen Resistance to MCF-7 Cells--
Because AKT
increases AF-1 activity and AF-1 is implicated in tamoxifen resistance,
we studied the 4-HT sensitivity of pcDNA3-1 and CA-AKT-4 cells
(Fig. 6A). Growth rates for
untreated, estrogen- (10
Reduced sensitivity of CA-AKT cells to 4-HT could be due to either loss
of 4-HT-induced cell cycle arrest at G0/G1 or
inhibition of 4-HT-induced apoptosis. To resolve this issue, we
measured the cell cycle distribution of untreated and 4-HT-treated
pcDNA3 and CA-AKT cells. Both pcDNA3 and CA-AKT cells
accumulated at G0/G1 phase at a similar rate
after 4-HT treatment (Fig. 6C). Apoptosis of untreated and
4-HT-treated (10 AKT Increases Bcl-2 but Not Cyclin D1 Expression--
Western blot
analysis of untreated and estrogen-treated cell extracts was performed
to identify estrogen-regulated cell cycle and anti-apoptotic genes that
are overexpressed in CA-AKT cells (Fig.
7). Cyclin D1 expression proved
estrogen-inducible in both cell types. AKT had no significant effect on
cyclin D1 expression. The known anti-apoptotic and estrogen-inducible
Bcl-2 gene (31) was expressed at very low levels and did not appear to
be up-regulated by estrogen in pcDNA3 cells. In CA-AKT-2 cells,
which expresses lower levels of AKT than CA-AKT-4 cells, estrogen
dramatically increased Bcl-2 expression. In CA-AKT-4 cells, Bcl-2 is
constitutively overexpressed. Expression of anti-apoptotic
Bcl-XL and pro-apoptotic BAK and BAX was similar in both
pcDNA3 and CA-AKT cells. These results suggest that AKT
specifically increases Bcl-2 expression in MCF-7 cells. Because Bcl-2
overexpression in CA-AKT-2 cells was observed only in the presence of
estrogen, it is likely that increased Bcl-2 expression in CA-AKT cells
requires ER In this report, we demonstrate that PI 3-kinase/AKT signaling
pathway modulates ER AF-1 region of ER We observed up-regulation of pS2, MIC-1, and Bcl-2 but not c-Myc in
CA-AKT-overexpressing cells (Fig. 5B). These results suggest that the ability of AKT to induce ERE-containing genes is promoter context-dependent. Because 4-HT repressed pS2 but not MIC-1
and Bcl-2 expression in CA-AKT cells, promoter context may also play a
role in determining the ability of 4-HT to suppress AKT-mediated activation of estrogen-responsive genes (Fig. 5B and data
not shown). Both pS2 and Bcl-2 are well characterized
estrogen-regulated genes, whereas MIC-1 promoter is yet to be
characterized. While induction of pS2 by estrogen requires an ERE (40),
activation of Bcl-2 by estrogen requires a SP-1 binding site, cAMP
response element-binding protein binding site, and an ERE in the Bcl-2 promoter (31, 41). Interestingly, cAMP response element-binding protein
binding site is required for AKT-mediated increase in Bcl-2 (42).
Therefore, it is likely that the ability of 4-HT to repress
AKT-mediated activation of ERE-containing promoters is determined by
promoter elements other than ERE. Alternatively, AKT may activate
certain ERE-containing promoters independent of ER Our results indicate that activation of the PI 3-kinase/AKT pathway
leads to increased Bcl-2 expression, which correlates with 4-HT
resistance in breast cancer cells. Consistent with our results,
Her2/Neu-mediated 4-HT resistance in MCF-7 correlates with Bcl-2
overexpression (45). However, the results of our cell culture studies
are inconsistent with clinical observations that correlate high Bcl-2
expression with favorable response to tamoxifen therapy and prolonged
disease-free intervals (46). It is possible that Bcl-2 works in concert
with additional anti-apoptotic proteins to confer tamoxifen resistance,
which is in accordance with the proposal that overall level of Bcl-2
family anti-apoptotic proteins dictates cellular response to therapy
(47).
The PI 3-kinase/AKT pathway is the major survival pathway for a wide
variety of cultured cell types (9). ER in the absence of estrogen. Although PI 3-kinase increased the activity of both
estrogen-independent activation function 1 (AF-1) and
estrogen-dependent activation function 2 (AF-2) of ER
, AKT
increased the activity of only AF-1. PTEN and a catalytically inactive
AKT decreased PI 3-kinase-induced AF-1 activity, suggesting that PI
3-kinase utilizes AKT-dependent and AKT-independent
pathways in activating ER
. The consensus AKT phosphorylation site
Ser-167 of ER
is required for phosphorylation and activation by AKT.
In addition, LY294002, a specific inhibitor of the PI 3-kinase/AKT
pathway, reduced phosphorylation of ER
in vivo.
Moreover, AKT overexpression led to up-regulation of estrogen-regulated
pS2 gene, Bcl-2, and macrophage inhibitory cytokine 1. We demonstrate
that AKT protects breast cancer cells from tamoxifen-induced apoptosis.
Taken together, these results define a molecular link between
activation of the PI 3-kinase/AKT survival pathways,
hormone-independent activation of ER
, and inhibition of
tamoxifen-induced apoptotic regression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
inhibits estrogen-stimulated growth of mammary epithelial cells.
Depending on the concentration of tamoxifen, growth-arrested cells
undergo apoptosis within 24 h or after 72 h of tamoxifen treatment (3). Although most ER
-positive breast cancers initially respond to tamoxifen therapy, tamoxifen-resistant tumors eventually develop (4). It has been shown previously that growth factors such as
epidermal growth factor (EGF), insulin-like growth factor (IGF-1), and
heregulin confer estrogen-independent growth properties to
ER
-positive breast cancer cells (5-7). It is suggested that EGF-
and IGF-1-induced mitogen-activated protein kinase (MAPK) phosphorylates Ser-118 of ER
, increases the activity of AF-1, and
confers hormone-independent growth (6). However, a recent study
indicates that, although prolonged activation of MAPK is growth
inhibitory in breast cancer cells, parallel activation of the PI
3-kinase/AKT pathway by EGF and IGF-1 is sufficient to overcome such
inhibition (8). Therefore, these growth factors may utilize the PI
3-kinase/AKT pathway to activate ER
and confer hormone-independent growth.
to the p85
regulatory subunit of PI 3-kinase (10). However, induction of AKT by
estrogen is cell type-specific, as it is not observed in MCF-7 breast
cancer cells (11). Activated AKT promotes cell survival by
phosphorylating and modulating the activity of various transcription
factors in the nucleus. The tumor suppressor PTEN gene, which
dephosphorylates 3'-phosphorylated phosphoinositides in
vivo, inhibits AKT activation (12). Several clinical and laboratory observations prompted us to study whether ER
is the target of AKT and whether AKT protects breast cancer cells against tamoxifen-induced apoptosis. For example, PI 3-kinase and AKT amplification is observed in breast and ovarian cancer (13-15). Cowden
disease patients, who have a germ line-inactivating PTEN mutation,
display increased breast cancer risks (16). Similarly, PTEN mutation is
observed in endometrial cancer (17). Moreover, IGF-1 and heregulin,
whose overexpression correlates with tamoxifen resistance, activate AKT
(5, 7). Finally, serine at amino acid position 167 of ER
is a
consensus AKT phosphorylation site (RXRXX(S/T))
(9). In this report, we show that AKT phosphorylates Ser-167 of ER
and confers tamoxifen resistance.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase expression vector RSV/
-galactosidase (2 µg) was cotransfected as
an internal control. In all transfections, the total amount of
expression vector was kept constant by substituting with appropriate empty expression vector. Fresh media (PR-free MEM + 5% CCS for MCF-7,
PR-free DMEM + 5% CCS for COS-1 cells) along with appropriate drugs
were added 24 h after transfection. Cells were harvested 36 h
after transfection and CAT activity in equal numbers of
-galactosidase units was determined as described previously (18).
ERE3-TK-CAT, GAL-AB and GAL-EF were gifts from P. Chambon and described
previously (19). The CA-AKT expression vector that lacks pleckstrin
homology domain (amino acids 4-129) of AKT and KD-AKT expression
vector that contains S179M substitution have been described previously (20) and were gifts from R. Roth. COS-1 cell transfections were performed with the original SV40 enhancer-promoter-driven CA-AKT and
KD-AKT expression vectors. pcDNA3 derivatives of CA-AKT and KD-AKT
were generated and used in MCF-7 cell transfections. Constitutively active PI 3-kinase vector, which contains murine p110
cDNA with avian Src myristoylation signal at the N terminus (21), was a gift from
S. Boswell. PTEN expression vector was a gift from J. Dixon.
were cloned in frame to pGEX-2T vector to obtain
recombinant GST-AB. AKT-kinase assay with recombinant GST, GST-AB, or
AB obtained after cleaving GST-AB with thrombin in the presence of
[
-32P]ATP was performed as per AKT manufacturer's
recommendations (Upstate Biotechnology, Inc.).
8
M estradiol or 10
6 M
4-HT or 10
7 M ICI-182,780 was
added and replaced every 48 h with appropriate drugs. At 72, 96, and 120 h, cells were collected by trypsinization, stained with
trypan blue, and counted twice using a hemocytometer. All experiments
were performed in triplicate.
6 M 4-HT. Cell cycle analysis
utilizing propidium iodide staining was performed at specific
intervals. Apoptosis of untreated and 4-HT-treated
(10
6 M for 72 h) control and
CA-AKT cells was measured by annexin V labeling and fluorescence
microscopy, as per manufacturer's recommendations (annexin V-EGFP, BioVision).
-tubulin (Sigma) was performed as per
manufacturer's recommendations. ER
antibodies were from either P. Chambon's laboratory or from Chemicon.
--
ER
cDNA was first cloned into pCMV4 expression
vector, which is similar to pcDNA3, except that it contains a
translational enhancer from alfalfa mosaic virus 4. 293 cells were
transfected with either empty expression vector or pCMV4-ER
. After
maintaining cells for 48 h in PR-free DMEM with 5% CCS, cells
were serum-starved in PR-free, phosphate-free DMEM for 24 h. Cells
were incubated with orthophosphate and either Me2SO or 20 µM LY294002. IGF-1 (100 ng/ml, R&D Systems) was added
after 1 h, and cells were harvested after an additional 1 h.
ER
was immunoprecipitated and subjected to autoradiography, followed
by Western blotting with ER
antibody.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
To
investigate the ability of AKT to directly regulate ER
activity,
ER
-positive MCF-7 breast cancer cells were transiently transfected
with a CAT reporter gene under the control of a thymidine kinase
promoter and three ERE elements (ERE3-TK-CAT). Estrogen increased
ERE3-TK-CAT activity 6-fold, whereas 4-hydroxytamoxifen (4-HT) had no
effect (Fig. 1A).
Cotransfection of a constitutively active AKT expression vector
(CA-AKT) resulted in 4.5- and 15-fold increases in ERE3-TK-CAT activity
in the absence and presence of estrogen, respectively (Fig.
1A). Cotransfection of a catalytically inactive AKT vector
(KD-AKT) affected neither estrogen-dependent nor
estrogen-independent ERE3-TK-CAT activity. These results suggest that
AKT confers estrogen-independent activity and further increases estrogen-stimulated activity of ER
. Note that AKT-mediated
estrogen-independent ERE3-TK-CAT activity was reduced to 2.5-fold
by 4-HT.
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Fig. 1.
AKT specifically increases
estrogen-independent activity of ER .
A, MCF-7 cells were transfected with 5 µg of ERE3-TK-CAT
and 1 µg of pcDNA3, CA-AKT, or KD-AKT. Cotransfection of AKT led
to increases in estrogen-independent activity and further increases in
estrogen-stimulated (E2, 10
8
M) activity of ER
. *, p = 0.0019; **,
p = <0.0001; and ***, p = 0.07 for
CA-AKT versus pcDNA3 in the absence and presence of
estrogen and 4-HT, respectively. Western blot analysis of extracts used
in a representative CAT assay for CA-AKT expression is shown.
B, PI 3-kinase-dependent increases in
ERE3-TK-CAT activity. Western blot of p85 subunit of PI 3-kinase that
coprecipitates with p110 subunit is also shown. *,
p = 0.0019 for untreated PI 3-kinase versus
untreated pcDNA3 cells. **, p = 0.0008 for
estrogen-treated PI 3-kinase versus estrogen-treated
pcDNA3 cells. ***, p = 0.0238 for 4-HT-treated PI
3-kinase cells versus 4-HT-treated pcDNA3 cells. C, AKT
had no effect on retinoic acid (10
6
M) inducible-expression of
-RARE-TK-CAT. In contrast, PI
3-kinase increased
-RARE-TK-CAT activity in the absence of retinoic
acid.
-RARE-TK-CAT (a TK promoter with a retinoic acid response element
from the
-retinoic acid receptor) (18). In contrast, PI 3-kinase
increased
-RARE-TK-CAT activity in the absence of retinoic acid
(Fig. 1C). These results suggest that, although AKT
overexpression leads to a specific increase in ER
activity, PI
3-kinase overexpression may lead to a global change in nuclear receptor
activity, possibly through activation of coactivators (2, 22).
required for estrogen-independent activation by AKT,
we utilized expression vectors that code for a fusion protein
containing the DNA binding domain (DBD) of yeast transcription factor
GAL-4 and either the AF-1 domain (GAL-AB) or AF-2 domain of ER
(GAL-EF) (19). A CAT reporter gene under the control of a TATA box and
five binding sites for GAL-4 DBD (pG5/CAT) was cotransfected with
either GAL(DBD) alone (pM), GAL-AB, or GAL-EF with or without CA-AKT.
COS-1 cells were used because they provide an ER
-free background.
CA-AKT increased GAL-AB activity by ~3.5-fold, which was not
influenced by estrogen (Fig. 2). GAL-EF
activity was observed only in estrogen-treated cells and was not
affected by AKT. These results demonstrate that AKT specifically
increases AF-1 activity and are consistent with previously described
EGF- and IGF-1-regulated enhancement of AF-1 activity (6).
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Fig. 2.
AF-1 is target for AKT. COS-1 cells were
transfected with GAL-DBD (pM), GAL-AB, or GAL-EF along with pG5/CAT and
RSV/ -galactosidase. GAL-AB activity in the absence of CA-AKT is set
arbitrarily as 10 units. GAL-AB activity increased by 3.5-fold in the
presence of CA-AKT. GAL-EF activity was not affected by AKT and was
appreciated only in estrogen-treated (10
8
M) cells. CA-AKT expression level in transfected cells is
shown.
and protein kinase C
,
which may contribute to a PI 3-kinase-mediated increase in ER
activity (23-25). To test this hypothesis, we transfected COS-1 cells
with pG5/CAT, GAL-AB, or GAL-EF and a constitutively active PI 3-kinase
(Fig. 3A). PI 3-kinase
increased GAL-AB activity by ~8-fold. Cotransfection with KD-AKT
resulted in reduction of PI 3-kinase-induced GAL-AB activity. PTEN, a
negative regulator of PI 3-kinase-mediated activation of AKT (12), also
showed a concentration-dependent reduction of PI
3-kinase-induced GAL-AB activity (Fig. 3B). For unknown
reasons, we observed reduced levels of PI 3-kinase p110·p85 complex
in cells transfected with 5 µg of PTEN (Fig. 3B,
bottom panel). Therefore, it is not clear whether reduction in GAL-AB activity by PTEN involves only its lipid
phosphatase activity or other biological activity. Nonetheless, PTEN
overexpression resulted in reduction in AKT activity in transfected
cells (data not shown). Interestingly, GAL-EF, which was not induced by
AKT (Fig. 2), was activated by PI 3-kinase both in the absence and presence of estrogen (Fig. 3A). Moreover, KD-AKT failed to
reduce GAL-EF activation by PI 3-kinase, suggesting that activation of AF-1 (but not AF-2) by PI 3-kinase is, at least in part,
AKT-dependent (Fig. 3A).
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Fig. 3.
PI 3-kinase activates AF-1 and AF-2 via
AKT-dependent and AKT-independent mechanisms,
respectively. COS-1 cells were transfected with the indicated
plasmids and pG5/CAT reporter. A, note that PI 3-kinase
increased both GAL-AB and GAL-EF activity. In addition, KD-AKT reduced
PI 3-kinase-induced GAL-AB activity, indicating that PI 3-kinase
utilizes AKT to activate AF-1. B, PTEN reduced PI
3-kinase-induced GAL-AB activity, suggesting that activation of AF-1 by
PI 3-kinase requires the presence of AKT. Expression levels of
endogenous and transfected genes are shown.
Is Essential for Phosphorylation and Activation by
AKT--
Phosphorylation of the AF-1 domain of ER
by AKT was
investigated using purified AKT and a recombinant protein containing the AF-1 domain fused to glutathione S-transferase
(GST-AB). GST and GST-AB proteins were incubated with
[
-32P]ATP and recombinant AKT. GST-AB but not GST was
phosphorylated by AKT, thereby confirming direct phosphorylation of
AF-1 by AKT (data not shown). Mass spectrometry followed by mutation
analysis revealed that AKT fortuitously phosphorylates Ser-173 or
Ser-178 of AF-1 within the context of GST-AB fusion protein (data not shown). Therefore, purified wild type AB or mutants lacking GST were
used in in vitro kinase assays. Mutation of Ser-167
(AB-S167A) but not Ser-118 (AB-S118A), Ser-173 (AB-S173A), Ser-178
(AB-A178A), and Ser-173/Ser-178 (AB-S173A,S178A) to alanine prevented
phosphorylation by AKT (Fig.
4A). Ser-167 is a consensus
AKT phosphorylation site (RXRXX(S/T)), which is
also phosphorylated by p90RSK1 (26), and is the major
phosphorylation site of ER
in MCF-7 cells (27).
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Fig. 4.
AKT directly phosphorylates AF-1.
A, phosphorylation of recombinant AB by purified AKT
in vitro. Recombinant GST, AB, or AB mutants were incubated
with recombinant AKT and [ -32P]ATP. Kinase reactions
were run on an SDS-PAGE, transferred to nitrocellulose, and subjected
to both autoradiography (top) and staining with Coomassie
Blue (bottom). B, effect of LY294002 on ER
phosphorylation. Orthophosphate-labeled ER
from 293 cells
transfected with either vector alone or pCMV4-ER
was identified by
immunoprecipitation and autoradiography (lanes
1-4). Western blot analysis of the same blot for ER
protein is also shown. Relative phosphorylation in each reaction was
calculated by densitometric scanning and normalizing for ER
level.
Orthophosphate-labeled proteins that coprecipitate with ER
are also
shown (lanes 5-8). NS, nonspecific.
C, serine 167 in the AB domain is essential for AKT-mediated
activation. COS-1 cells were transfected with either GAL-AB or mutants
as in Fig. 2. AB-S167A mutant in which serine 167 is mutated to alanine
was not activated by CA-AKT. Wild type and mutant GAL-AB proteins are
expressed at a similar level in transfected cells (data not
shown).
is dependent on PI 3-kinase/AKT pathway. Human embryonic kidney
cells 293 transfected with ER
expression vector were labeled with
[32P]orthophosphate and treated with Me2SO,
IGF-1, or LY294002 + IGF-1. LY294002 is a specific inhibitor of PI
3-kinase/AKT pathway. Although IGF-1 treatment led to only marginal
increase in overall ER
phosphorylation, LY294002 inhibited ER
phosphorylation (Fig. 4B). The effect of LY294002 is
specific to ER
, as phosphorylation of an unknown protein that
coprecipitates with ER
was similar in both untreated and
LY294002-treated cells (Fig. 4B, lanes
5-8, indicated by **). These results further suggest the
involvement of PI 3-kinase/AKT pathway in phosphorylation of ER
.
in COS-1, MDA-MB-231, and HeLa cells (data not
shown). AKT-mediated activation of AB-S102N, S104P, S106A, and AB-S118A
was also lower than the wild type, which could be due to reduced basal
activity of these mutants. Alternatively, activation of AF-1 by AKT is
partially dependent on phosphorylation of AB by cdk2/cyclinA or MAPK
(6, 28). In this regard, we have observed that mutation of both cdk2/cyclin A and AKT phosphorylation sites severely compromised ligand-independent activity of full-length ER
in MDA-MB-231
cells.2
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Fig. 5.
AKT increases pS2 and MIC-1 expression.
A, MCF-7 cells stably overexpressing AKT (CA-AKT-2,
CA-AKT-4) and empty vector (pcDNA3-1, pcDNA3-2) were
developed and analyzed for CA-AKT expression by Western blotting.
Endogenous AKT and transfected constitutively active AKT (CA-AKT) are
indicated. B, AKT increases pS2 but not c-Myc expression.
Total RNA (20 µg) from cells grown in PR-free MEM + CCS with or
without treatment for 1 h was subjected to Northern analysis with
indicated probes. The integrity of RNA was verified by reprobing the
blot with 36B4 ribosomal protein gene cDNA.
8 M),
4-HT- (10
6 M), and
estrogen+4-HT-treated cells were determined at specific intervals. As
expected, estrogen stimulated the growth of both pcDNA3 and CA-AKT
cells. Also note that we reproducibly observed ~20% increase in the
basal growth rate of untreated CA-AKT compared with untreated
pcDNA3 cells. Significantly, 4-HT was more efficient in inhibiting
the growth of pcDNA3 compared with CA-AKT cells, indicating that
AKT reduces 4-HT sensitivity of MCF-7 cells. In contrast to 4-HT,
CA-AKT cells were significantly growth-inhibited by the pure
anti-estrogen ICI-182,780 (Fig. 6B), which inhibits ER
activity by preventing DNA binding and enhancing degradation (30).
These results suggest that modulation of ER
activity by AKT is
responsible for 4-HT resistance of CA-AKT cells, although we can not
completely rule out the involvement of ER
-independent anti-apoptotic
activity of AKT in 4-HT resistance.
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Fig. 6.
AKT overexpression confers tamoxifen
resistance through inhibition of tamoxifen-induced apoptosis.
A, growth rates of untreated and estrogen-treated
(10 8 M) and/or 4-HT-treated
(10
6 M) pcDNA3-1 and
CA-AKT-4 cells were measured by trypan blue exclusion and manual
counting at specific intervals. CA-AKT cells were significantly more
resistant to 4-HT than pcDNA3 cells. *, p < 0.0004 for 4-HT-treated pcDNA3 versus 4-HT-treated CA-AKT cells
(day 5). B, growth rates of untreated and estrogen-treated
(10
8 M) or ICI-182,780-treated
(10
7 M) pcDNA3-1 and
CA-AKT-4 cells. **, p = 0.0001 for untreated
versus ICI-treated pcDNA3 cells. **, p = 0.0078 for untreated versus ICI-treated CA-AKT-4 cells.
C, pcDNA3 and CA-AKT cells arrest at
G0/G1 phase at a similar rate after 4-HT
treatment. Cell cycle distribution was determined by flow cytometric
analysis of propidium iodide-stained cells. D, AKT inhibits
4-HT-induced apoptosis. Cells were treated with 4-HT
(10
6 M) for 72 h, and rate
of apoptosis was measured by annexin V labeling. **, p < 0.0001 for percentage of apoptosis in 4-HT-treated CA-AKT
versus 4-HT-treated pcDNA3 cells. ***, p = 0.59 for untreated versus 4-HT-treated CA-AKT
cells
6 M for 72 h) pcDNA3 and CA-AKT cells were measured by annexin V labeling and
fluorescence microscopy (Fig. 6D). A significantly higher
percentage of pcDNA3 cells (31%) were apoptotic when compared with
CA-AKT cells (12%) after 4-HT treatment. AKT therefore inhibits 4-HT-induced apoptosis but not cell cycle arrest of MCF-7 cells.
activity.
View larger version (20K):
[in a new window]
Fig. 7.
AKT up-regulates Bcl-2. Whole cell
extracts from untreated and estrogen-treated
(10 8 M) pcDNA3 and CA-AKT
cells were subjected to Western analysis using the indicated
antibodies. AKT overexpression led to specific up-regulation of the
anti-apoptotic Bcl-2 gene.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
activity in vivo, which correlates
with phosphorylation of Ser-167 by AKT in vitro. Recent
studies indicate that estrogen promotes association of ER
with IGF-1
receptor and p85 subunit of PI 3-kinase in the plasma membrane, which
leads to AKT activation (10, 32). Our results suggest that AKT serves as a functional link between membrane-associated and nuclear ER
. By
phosphorylating Ser-167, AKT may modulate coactivator:AF-1 and/or
corepressor:AF-1 interactions in the nucleus. Consistent with this
possibility, previous studies have shown that phosphorylation of
Ser-118 of ER
alters both coactivator:AF-1 and corepressor:AF-1 interactions (33, 34).
contains phosphorylation sites for a number of
kinases including MAPK and cyclin A/cdk2 (6, 28, 35). Some of these
sites are conserved between ER
and ER
(36). However, AKT
phosphorylation site is present only in ER
(36). ERs exist as
homodimers as well as ER
·ER
heterodimers, and these combinations have different affinity for EREs (37, 38). Therefore, induction of ERE containing genes upon activation of AKT may be cell
type-dependent. Furthermore, regulation of ER
activity
by AKT may be controlled by p90RSK1 as it can also
phosphorylate Ser-167 (26). Finally, MAPK may indirectly control
AKT-mediated activation of ER
as it can activate p90RSK1
(39).
, which is not
subject to repression by 4-HT. Additional experiments with dominant
negative mutants of ER
(43, 44) are essential to distinguish between
these possibilities.
may be the central element in
this survival pathway, at least in certain cell types. Broader
implications of our results are on endometrial cancers as well as on
Cowden's disease patients, who show a 30-50% incidence of breast
cancer in affected females (16). In both neoplastic conditions, AKT
activity is increased due to mutation of PTEN. The PI 3-kinase/AKT
pathway, therefore, may be an ideal target for therapeutic intervention
in these cancers.
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ACKNOWLEDGEMENTS |
---|
We thank P. Chambon, S. Boswell, J. Dixon, D. Donner, R. Roth, and Y. C. Yang for various plasmids. We also thank J. Hawes for mass spectrometry and S. Rice for flow cytometry.
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FOOTNOTES |
---|
* This work was supported by the Catherine Peachy Fund, Inc. and by American Cancer Society Grant RPG-00-122-01-TBE (to H. N.).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.
** To whom correspondence should be addressed: R4-202, Indiana Cancer Research Inst., 1044 W. Walnut St., Indianapolis, IN 46202. Tel.: 317-278-2238; Fax: 317-274-0396; E-mail: hnakshat@iupui.edu.
Published, JBC Papers in Press, January 3, 2001, DOI 10.1074/jbc.M010840200
2 H. Nakshatri, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are: ER, estrogen receptor; ERE, estrogen response element; 4-HT, 4-hydroxytamoxifen; IGF, insulin-like growth factor; MIC-1, macrophage inhibitory cytokine-1; AF, activation function; EGF, epidermal growth factor; MEM, minimal essential medium; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; MAPK, mitogen-activated protein kinase; PI, phosphatidylinositol; PR, phenol red; CAT, chloramphenicol acetyltransferase; GST, glutathione S-transferase; TK, thymidine kinase; RARE, retinoic acid response element; DBD, DNA binding domain; CCS, charcoal/dextran-treated serum; AB, amino-terminal A/B region.
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