Enhancement of Insulin-Like Growth Factor Signaling in Human Breast Cancer: Estrogen Regulation of Insulin Receptor Substrate-1 Expression in Vitro and in Vivo
Adrian V. Lee1,
James G. Jackson1,
Jennifer L. Gooch,
Susan G. Hilsenbeck,
Ester Coronado-Heinsohn,
C. Kent Osborne and
Douglas Yee
Department of Medicine Division of Medical Oncology
University of Texas Health Science Center at San Antonio San
Antonio, Texas 78284-7884
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ABSTRACT
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Cross-talk between insulin-like growth factor
(IGF)- and estrogen receptor (ER)-signaling pathways results in
synergistic growth. We show here that estrogen enhances IGF signaling
by inducing expression of three key IGF- regulatory molecules, the type
1 IGF receptor (IGFR1) and its downstream signaling molecules, insulin
receptor substrate (IRS)-1 and IRS-2. Estrogen induction of IGFR1 and
IRS expression resulted in enhanced tyrosine phosphorylation of IRS-1
after IGF-I stimulation, followed by enhanced mitogen-activated protein
kinase activation. To examine whether these pathways were
similarly activated in vivo, we examined MCF-7 cells grown
as xenografts in athymic mice. IRS-1 was expressed at high levels in
estrogen-dependent growth of MCF-7 xenografts, but withdrawal of
estrogen, which decreased tumor growth, resulted in a dramatic decrease
in IRS-1 expression. Finally, we have shown that high IRS-1 expression
is an indicator of early disease recurrence in ER-positive human
primary breast tumors. Taken together, these data not only reinforce
the concept of cross-talk between IGF- and ER-sig-naling pathways,
but indicate that IGF molecules may be critical regulators of
estrogen-mediated growth and breast cancer pathogenesis.
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INTRODUCTION
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One of the most important predictive and prognostic markers in
human breast cancer is the estrogen receptor (ER). Long before the
receptor was cloned, or its function understood, antihormonal therapies
were used to treat breast cancer, and long-term studies have since
shown the importance of antiestrogens such as tamoxifen (Tam) in
reducing both disease progression and contralateral breast cancer (1).
Due to the proliferative action of estrogen in vitro, much
research has examined estrogens ability to control proliferation via
regulation of autocrine and paracrine growth factors (2). From these
studies data have emerged supporting a role for insulin-like growth
factors (IGFs) in estrogen action from in vitro, in
vivo, and clinical studies (3).
The IGF family plays an important role in the growth of both
normal and neoplastic cells (4). Gene-knockout studies have revealed
the complexity of IGF signaling and have shown that IGFs can have
mitogenic, transforming (probably via a permissive action), and
antiapoptotic functions (5). Examination of IGF expression in primary
human breast tumors has shown that at all stages of IGF action,
expression of the components of the effector pathways are correlated
with ER expression and have prognostic significance. For instance,
IGF-binding protein (IGFBP) expression in primary human breast tumors
is correlated with ER status, and high IGFBP-3 and IGFBP-4 levels are
associated with poor prognostic markers (6, 7). The type 1 IGF receptor
(IGFR1) is expressed in a high percentage of primary breast tumors, and
expression is positively correlated with ER status (8). One of the
downstream signaling molecules of IGFR1, insulin receptor substrate 1
(IRS-1), is expressed in human breast cancer, and a high level of
expression is an indicator of early disease recurrence in small tumors
(7).
Animal studies support a role for IGFs in the pathogenesis of breast
cancer. MDA-231 xenograft tumor growth can be inhibited by blockade of
IGFR1 with the monoclonal antibody
IR3 (9). We followed this
observation by showing that neutralization of IGF action with IGFBP-1
inhibited growth of MDA-231 and also ascites growth of MDA-435A (10).
More recent evidence that endocrine IGFs are important in breast tumor
growth is shown by the reduced growth of MCF-7 xenografts in mice that
lack circulating IGF-I (11). Taken together, these data show that
circulating IGFs are important in these xenograft models of breast
cancer. Clinically, this is important, considering the ability of Tam
to consistently reduce serum IGF-I levels in breast cancer patients
(12).
Strengthening the clinical and animal studies that support a role for
IGFs in breast cancer pathogenesis are a number of in vitro
studies that not only confirm the potent mitogenic effects of IGFs on
breast cancer cells, but also highlight considerable synergism between
IGF and ER signaling. Estrogen can affect IGF action and growth by
altering expression of IGFR1 (13 13A ), IGFR2 (14), IGF-II (15), and
IGFBPs (16, 17). It is not surprising, therefore, that overexpression
of certain IGF family members (IGF-II and IRS-1) results in enhanced
growth and reduced estrogen requirements (18, 19, 20, 21, 22). Conversely,
decreased expression of IRS-1 inhibits breast cancer cell proliferation
and causes cell death in serum-free conditions (23).
In an attempt to understand cross-talk and synergism between IGF and ER
signaling, we examined regulation of IGF family members by estrogen and
antiestrogens. We present data here confirming that IGFR1 is an
estrogen-regulated protein, but we also show that expression of the
major downstream targets of IGFR1, IRS-1, and IRS-2 are regulated by
estrogen. Increased expression of IGFR1, IRS-1, and IRS-2 by estrogen
results in enhanced IRS phosphorylation, which leads to greater
mitogen-activated protein kinase (MAPK) activity. Furthermore, we have
shown that IRS-1 is regulated by estrogen in a xenograft model of human
breast cancer, and that in ER-positive human breast tumors high IRS-1
expression is associated with poor disease-free survival (DFS). Taken
together, these data support a role for IGFs in ER-mediated growth,
with components of IGF signaling pathways being key targets for ER
action and thus, in part, responsible for estrogens growth promoting
effects.
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RESULTS
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Enhancement of MCF-7 Cell Growth by Coincubation with Estrogen and
IGF-I
MCF-7 cells were incubated with estrogen (1 nM), IGF-I
(5 nM), or a combination for 5 days, and cell number was
counted. Estrogen and IGF-I treatment resulted in a 2.2-fold increase
in cell number compared with cells incubated in serum-free medium (SFM)
(Fig. 1
). Coincubation with both mitogens
resulted in greater proliferation and a 5-fold increase in cell number.
This result was seen consistently, with the effect of estrogen and
IGF-I being greater than estrogen or IGF-I alone.

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Figure 1. Enhancement of MCF-7 Cell Growth by Coincubation
with Estrogen and IGF-I
MCF-7 cells were incubated in SFM, E2 (1
nM), IGF-I (5 nM), or a combination for 5 days,
and then cell number was counted by hemocytometer. Bars represent the
average of triplicate cells ± SEM. This figure is
representative of three independent experiments.
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Estrogen Increases IRS-1 Expression in ER-Positive Breast Cancer
Cell Lines
Three ER-positive human breast cancer cell lines (MCF-7, T47D, and
ZR-75) and an ER-negative breast cancer cell line (MDA-435A) were
incubated with or without estrogen for increasing lengths of time (2,
4, and 6 days), and extracts were immunoblotted for IRS-1 expression
(Fig. 2
). Estrogen increased IRS-1
expression in all three ER-positive cell lines, but had no effect in
MDA-435A cells. A consistent decrease in IRS-1 expression was seen in
all ER-positive cells grown in SFM, probably representing depletion of
residual estrogen. Further experiments did indeed show that
antiestrogens reduced this basal IRS-1 expression (data not shown and
next figure).

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Figure 2. Estrogen Increases IRS-1 Expression in ER-Positive
Breast Cancer Cell Lines
Three ER-positive (MCF-7, T47D, and ZR-75) and one ER-negative
(MDA-435A) breast cancer cell line were stimulated with or without
E2 (1 nM) for increasing periods of time (2, 4,
and 6 days). Cells were lysed and immunoblotted for IRS-1. This result
is representative of two independent experiments.
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Expression of IRS-1, IRS-2, and IGFR1 Is Regulated by Estrogen
MCF-7 cells were incubated with estrogen and the pure steroidal
antiestrogen ICI 182780 (ICI) for increasing lengths of time (8, 24,
and 48 h) and analyzed for IGFR1, IRS-1, and IRS-2 expression.
IGFR1 mRNA- and IGF-I-binding sites have previously been shown to be
up-regulated after estrogen treatment of breast and endometrial cancer
cells (13 13A, 24). Confirming this observation, we saw that after
48 h, estrogen increased IGFR1 protein expression (Fig. 3
), and that this stimulation was
specifically competed by ICI. No difference in IGFR1 expression was
seen at 8 and 24 h of estrogen treatment. Expression of IRS-1 and
IRS-2 was also increased by estrogen and specifically inhibited by ICI.
In some instances, IRS expression in the presence of antiestrogen was
actually reduced below that of cells in SFM [compare IRS-1 expression
at 48 h SFM vs. estradiol
(E2)+ICI], again suggesting that there is residual
estrogen present. However, in contrast to the relatively late induction
of IGFR1, IRS-1 and IRS-2 levels were also increased at earlier time
points (8 and 24 h). Up-regulation of IGFR1 and IRS expression was
not simply due to the fact that cells were proliferating faster in the
presence of estrogen, since growth stimulation by IGF-I treatment
resulted in no change in IGFR1 expression and an actual decrease in
IRS-1 expression (data not shown). Additionally we have shown that IRS1
expression is decreased during S-phase (data not shown), ruling out the
possibility that estrogen induction of IRS-1 is simply due to the fact
that cells are proliferating faster and that more cells are in
S-phase.

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Figure 3. Estrogen Increases Expression of IGFR1, IRS-1, and
IRS-2 in MCF-7 Cells
MCF-7 cells were stimulated with E2 (1 nM) and
ICI (1 µM) alone or in combination for increasing periods
of time (8, 24, and 48 h). Cells were lysed and immunoblotted for
IGFR1, IRS-1, and IRS-2. Results shown are representative of four
independent experiments.
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Estrogen Increases Expression of IGFR1 and IRS-1 mRNA
MCF-7 cells were treated with estrogen and antiestrogen for 24 and
48 h, total RNA was isolated, and mRNA expression was examined by
RNAse protection assay. Estrogen resulted in increased mRNA expression
of both IRS-1 and IGFR1 at both time points (Fig. 4A
). Additionally, both antiestrogens,
Tam and ICI, effectively reversed the induction. The expression is
represented graphically after correcting for the loading control (Fig. 4B
). The lower induction of IRS-1 expression at 48 h represents
experimental variation and was not seen in all experiments. As a
control, we analyzed IGFR1 and IRS-1 mRNA expression after exposure of
cells to IGF-I. In this instance there was no change in either mRNA
(data not shown), indicating again that estrogen-induction is not
simply a result of estrogen-stimulated proliferation.

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Figure 4. Estrogen Increases IGFR1 and IRS-1 mRNA Expression
in MCF-7 Cells
A, MCF-7 cells were stimulated with E2 (1 nM),
Tam (1 µM), or ICI (1 µM), alone or in
combination for 24 or 48 h. RNA was prepared and 20 µg analyzed
by RNAse protection assay. 36B4 was used as a loading control. B,
Graphical representation of data in panel A after densitometry and
correction for 36B4 expression. This figure is representative of three
independent RNAse protection assays. C, MCF-7 cells were
pretreated with (Act D) or without (SFM) actinomycin D (2 µg/ml) for
30 min and then stimulated with E2 (1 nM) in
the absence or presence of actinomycin D for 24 h. Cells were
lysed and immunoblotted for IRS-1 and IGFR1.
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Having shown that IRS-1 mRNA and protein expression was induced
relatively quickly after estrogen stimulation, we examined whether this
was a transcriptional effect. MCF-7 cells were stimulated with estrogen
in the presence or absence of actinomycin D (2 µg/ml). The induction
of IGFR1 and IRS-1 was completely blocked by actinomycin D (Fig. 4C
),
suggesting that estrogen induces IGFR1 and IRS-1 mRNA by a
transcriptional mechanism. However, there was a slight discordance
between the level of estrogen induction of IGFR1 protein (Fig. 3
) and
IGFR1 mRNA (Fig. 4A
), suggesting that there may be nontranscriptional
mechanisms of regulation.
Estrogen Increases IGF Signal Transduction Events
We sought to examine the effect of altering IRS-1 and IGFR1 levels
on IGF signal transduction by first preincubating cells with estrogen,
or with estrogen and ICI for 48 h, and then stimulating with IGF-I
for 10 min. As expected, estrogen increased expression of IRS-1 and
IGFR1, which was blocked by ICI (Fig. 5
, panels 2 and 3). Treatment of cells with IGF-I for 10 min resulted in
tyrosine phosphorylation (PY) of a single protein of
approximately 180 kDa (panel 1). We have recently shown by
immunoprecipitation that this tyrosine-phosphorylated protein is IRS-1,
and that while MCF-7 and other ER-positive breast cancer cells express
IRS-2, that IRS-2 is not activated in these cells (25). After exposure
to estrogen, IGF-I stimulation resulted in enhanced phosphorylation of
IRS-1 compared with cells stimulated with IGF-I but without estrogen
pretreatment. The increased phosphorylation of IRS-1 generally mirrored
the increase in IRS-1 protein seen by immunoblotting. Preincubation of
cells with estrogen and ICI resulted in reduced IRS phosphorylation
compared with cells in estrogen.

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Figure 5. Estrogen Increases IGF Action in MCF-7 Cells
MCF-7 cells stimulated with E2 (1 nM) or
E2 and ICI (1 µM) for 48 h (48 h). At
the end of the 48 h, cells were stimulated with IGF-I (5
nM) for 10 min (10 m). Cells were lysed and immunoblotted
with antibodies to phosphotyrosine (PY, panel 1), IRS-1 (panel 2),
IGFR1 (panel 3), phospho-specific MAPK (panel 4), or total MAPK protein
(panel 5). The figure is representative of three independent
experiments.
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To examine whether estrogen enhancement of IRS expression and
phosphorylation resulted in increased downstream signaling, we measured
activation of MAPK using a phospho-specific MAPK antibody. We have
previously determined in our laboratory that there is good concordance
between the phosphospecific MAPK antibodies and actual measurement of
MAPK activity (measured by the ability to phosphorylate myelin basic
protein). Estrogen enhancement of IRS-1 phosphorylation resulted in an
increase in IGF-I-induced phospho-MAPK (panel 4). Densitometric
analysis revealed that IGF induction of MAPK activity in the presence
of estrogen was 5-fold higher than IGF activation of MAPK in cells
grown in SFM. Again, this increase in MAPK activity was inhibited by
ICI. A similar pattern of estrogen induction of IRS phosphorylation and
MAPK activity was seen at shorter times of estrogen treatment (8 and
24 h; data not shown) although the absolute level of induction was
smaller than that seen at 48 h. Total levels of MAPK protein were
not affected under any of the conditions tested (panel 5).
IRS-1 Expression and Phosphorylation Are Estrogen Regulated in an
in Vivo Model of Human Breast Cancer
As we have previously shown that IRS-1, and not IRS-2, is the
major downstream signaling molecule in MCF-7 cells (25), we further
investigated regulation of IRS-1 expression in vivo in a
xenograft model of human breast cancer growth. We examined tumors grown
in the presence or absence of estrogen for both total IRS-1 protein
expression and tyrosine phosphorylation of IRS-1. Similar to cells
grown in vitro, tumors growing in the presence of estrogen
(+E2) had high levels of IRS-1 expression (Fig. 6A
, second panel). When the estrogen
pellet was removed (-E2), there was a dramatic reduction
in IRS-1 expression. MCF-7 cells grown in vitro were used as
a positive control. In two independent experiments eight of eight
tumors grown in the presence of estrogen expressed high levels of
IRS-1. When estrogen was removed, there was a greater than 95%
reduction of IRS-1 expression in 22 of 23 tumors. We then performed
antiphosphotyrosine analysis on tumors growing in the presence or
absence of estrogen, which revealed an immunoreactive species of the
same mol wt as IRS-1 (Fig. 6A
, top panel).
Immunoprecipitation with IRS-1 antibodies followed by
antiphosphotyrosine immunoblotting revealed a band that comigrated with
IRS-1 immunoprecipitated from MCF-7 cells grown in vitro,
indicating that this was indeed tyrosine-phosphorylated IRS-1 (Fig. 6B
). Immunoprecipitation from two individual xenografts grown in the
presence of estrogen showed tyrosine-phosphorylated IRS-1 whereas we
detected absolutely no tyrosine-phosphorylated IRS-1 in xenografts
grown in the absence of estrogen.

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Figure 6. IRS-1 Expression and Activation Are Regulated by
Estrogen in MCF-7 Xenograft Tumors
MCF-7 cells were grown as flank tumors in nude mice. A, Four individual
MCF-7 xenografts were harvested either when growing in the presence of
estrogen (+E2), or when estrogen was removed
(-E2). Tumors were crushed under liquid nitrogen, lysed,
and immunoblotted for phosphotyrosine (PY), IRS-1, active MAPK, and
total MAPK. MCF-7 cells grown in vitro stimulated with
IGF-I for 10 min were used as a positive control (MCF7L). This figure
shows representative samples of eight tumors +E2 and 23
tumors -E2. B, Individual xenograft tumors from Fig. 6A
that were grown in the presence (+E2, tumors 1 and 2) or
absence of estrogen (-E2, tumors 5 and 6) were
immunoprecipitated with antibodies to IRS-1 and immunoblotted with an
antibody to PY.
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In vitro data from Fig. 5
, panel 4, and the presence of
tyrosine- phosphorylated IRS-1 in estrogen-treated tumors suggested
that IRS-1 was in an active signaling cascade; therefore, we analyzed
activation of downstream MAPK in the same tumors. The tumors that were
growing in the presence of estrogen (+E2) and had high
levels of tyrosine-phosphorylated IRS-1 (second panel) also had high
levels of phosphorylated MAPK (Fig. 6A
, third panel). In absolute
contrast, removal of estrogen (-E2), which resulted in
loss of IRS-1 tyrosine phosphorylation and expression, resulted in
complete loss of MAPK activity. Immunoblotting for total MAPK levels
indicated that there was no change in total MAPK expression among the
tumors and served as loading control. While there are many upstream
activators of MAPK, in all samples we saw an absolute correlation
between IRS-1 expression, tyrosine phosphorylation, and MAPK activity
(data not shown).
IRS-1 Expression Affects Breast Cancer Recurrence in ER-Positive
Breast Cancer Patients
Since our in vitro and xenograft studies show that ER
and IGF may act synergistically to enhance growth, we reasoned that
tumors expressing both ER and IRS-1 would have a growth advantage
reflected by early recurrence after surgery. To test this hypothesis,
we examined the influence of IRS-1 expression on DFS in ER-positive
breast cancer patients. We have previously reported that IRS-1
expression positively correlates with ER levels (7). Indeed, after
cutpoint analysis, breast tumor samples were separated into high or low
IRS-1 expression by median IRS-1 levels, and nearly all of the
ER-negative tumors had low IRS-1 expression. Furthermore, in the subset
of ER-positive patients, patients with high IRS-1 expression had
significantly shortened DFS (P = 0.035) than patients
with low IRS-1 levels (Fig. 7
). In the
ER-negative subset of patients, IRS-1 levels had no effect upon DFS;
however, the low number of samples did not provide enough data to reach
statistical significance. The fact that ER-positive tumors with high
IRS-1 expression have poor DFS suggests that the relationship between
ER and IRS-1 that we have observed in vitro and in xenograft
models is also relevant in human primary breast cancers.

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Figure 7. High IRS-1 Expression Is Associated with Reduced
DFS in ER-Positive Breast Cancer Patients
Patients with lower IRS-1 levels (solid line, n =
48) had significantly better DFS (P = 0.035 by log
rank test) than patients with higher IRS-1 levels (dotted
line, n = 99). The cutoff used to separate tumors
expressing high levels of IRS-1 was the median of all samples analyzed.
For graphing, follow up is truncated at 10 yr.
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DISCUSSION
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While the potent mitogenic effect of estrogen has been known for a
long time, the mechanism of estrogen-mediated proliferation remains
unclear. Estrogen acts through a nuclear hormone receptor, which upon
activation can induce transcription of hormone-responsive genes. Many
candidates were initially found to be involved in mitogenesis,
including DNA-synthesizing enzymes, other hormone receptors, and
autocrine and paracrine growth factors and their receptors (26).
Further studies showing that overexpression of some of these growth
factors and receptors caused estrogen-independent growth (19, 20) led
to the "autocrine hypothesis": estrogen stimulation of cells
results in enhanced growth factor secretion, which then acts upon the
same cells to stimulate proliferation (27). However, it is clear from
recent studies that estrogen can directly affect the cell-cycle
machinery before increased growth factor secretion is observed
(28, 29, 30), suggesting that estrogen-mediated growth probably represents
a combination of an early direct effect upon the cell cycle machinery
and a late effect upon growth factor signaling.
Previous reports have shown that estrogen may regulate IGF activity by
altering expression of IGFBPs and IGFR1 mRNA and IGF-binding sites (13 13A, 24). We confirm reports of estrogen induction of IGFR1 expression
but also show for the first time that estrogen can induce expression of
the downstream signaling molecules, IRS-1 and IRS-2. Estrogen induction
of IRS-1 expression was associated with increased tyrosine
phosphorylation of IRS-1 after IGF stimulation and correlated with
enhanced downstream MAPK activation. Thus, efficacy of IGF signaling,
i.e. the maximum achievable activity, was increased. We
would predict that other IGF-signaling pathways will be similarly
affected and these are under investigation. While the increase in IRS-1
expression generally mirrored the increase in tyrosine phosphorylation,
we cannot rule out the possibility that the increase in tyrosine
phosphorylation of IRS-1 results from a change in stoichiometry or
sites of phosphorylation. Additionally, there may be other factors
controlling phosphorylation of IRS-1. For instance, decreased
phosphorylation of IRS may be mediated by antiestrogen-induction of a
specific tyrosine phosphatase activity, which has been proposed
previously by other groups (22, 31, 32).
Synergism between estrogen and IGF has been shown in a number of model
systems including normal breast (33), normal uterus (34), endometrial
cancer cells (24), and breast cancer cells (13). In many of these
systems, and in the data we present here, cotreatment with estrogen and
IGF-I causes growth and signaling that is greater than IGF-I alone.
While we hypothesize that estrogen actually sensitized cells to IGFs by
up-regulating expression of IGF-signaling components, testing this
hypothesis in growth assays is problematic due to the potential of
IGFBPs to influence low levels of IGF interaction with the receptor,
and the fact that expression of the IGFBPs are regulated by estrogen.
However, our data showing enhanced growth with E2
and IGF-I compared with either ligand alone is completely consistent
with the observation that increased levels of IRS-1 achieved by
transfection result in enhanced growth of these cells and decreased
requirements for estrogen (35).
It has recently been suggested that the main mechanism of
estrogen-mediated growth is through early activation of
cyclin-dependent kinases (Cdk-2 and Cdk-4), phosphorylation of pRB, and
increased expression of cyclins within 28 h of hormone stimulation
(28, 29, 30). As changes in growth factors tend to occur at later time
points, their importance in estrogen-mediated growth has been
challenged (29). However, the short time for induction of IRS
expression and activation (8 h), similar to the recently reported early
estrogen induction of tyrosine phosphorylation of IGFR1 and IRS-1 in
the rat uterus (34), suggests that early-growth factor signaling
activation may be an important component in estrogen-mediated growth.
While regulation of cell-cycle regulatory components is probably
sufficient to send cells through one round of the cell cycle, it is
probably not sufficient for maximum estrogen-mediated growth. There
must be, in addition, some level of growth factor signaling to allow
cells to pass the restriction point of the cell cycle (36). We believe
that the early induction of IRS-1, which has been shown to be a crucial
rate-limiting step in IGF signal transduction in MCF-7 cells (18, 23),
may "sensitize" cells to autocrine, paracrine, or endocrine sources
of IGFs, which results in early activation of IGF signaling that allows
cells to pass the restriction point. In contrast to the autocrine
hypothesis (27, 37), estrogen treatment of cells increases expression
of IGF signaling components within the cell and thus enhances
sensitivity to any source of IGFs. After the initial entry into and
movement through the G1 phase of the cell cycle, estrogen
then causes a relatively late (2448 h) induction of other
growth-signaling proteins, e.g. IGFR1 and IGF-II (38), which
then act in an autocrine or paracrine manner and cause synergistic and
maximal proliferation.
While our studies indicate that estrogen induces IGFR1 and IRS-1
expression by a transcriptional mechanism, we do not know whether this
is a direct effect of ER upon either promoter. For instance, estrogen
induction of cyclin D1 expression probably does not result from a
direct effect of ER upon the promoter, but rather from estrogen
induction of other cis-acting factors such as
c-myc or AP-1, which are both increased by estrogen (39, 40)
and have been shown to increase cyclin D1 mRNA expression (30, 41). The
IRS-1 promoter does have four consensus half-estrogen response
elements, supporting the possibility of direct ER regulation, probably
through synergism between multiple ERs (42). Furthermore, the IRS-1
promoter contains several AP-1 and SP-1 sites (43), which in other
promoters have been shown to interact with the ER and activate
transcription in a synergistic manner (44, 45).
Consistent with estrogen regulation of IRS-1 expression in
vitro, IRS-1 expression is regulated in the MCF-7 xenograft model
of breast cancer. In the presence of estrogen, and when the tumor is
growing exponentially, IRS-1 is both expressed at high levels and
tyrosine phosphorylated and is thus presumably involved in an active
mitogenic signal transduction cascade. Indeed, active IRS-1 is
associated with downstream MAPK activity. These data support previous
work showing that estrogen increases tyrosine phosphorylation of IGFR1
and IRS-1 in the epithelial layer of the rat uterus (34). We do not
know at present the factors responsible for the activation of IRS-1 in
the MCF-7 xenograft. It may be a result of 1) autocrine or paracrine
IGF expression from within the xenograft, 2) endocrine IGF-I from the
host mouse, or 3) an unrelated IGF event, since IRS molecules are
involved in signaling by several cytokines and other ligands (46).
Interestingly, growth of MCF-7 xenograft tumors is retarded in mice
lacking circulating IGF-I (11), suggesting that endocrine IGF-I may
affect the proliferation of these cells. Removal of estrogen resulted
in no detectable IRS-1, as was observed in vitro.
Considering the evidence indicating that ER regulates IRS-1 expression
in vitro and in vivo, and after our initial
observation that high IRS-1 expression is a poor prognostic indicator
in small tumors (7), we reanalyzed the same data set of node-negative
patients to determine whether IRS-1 levels have prognostic significance
in ER-positive tumors. Analysis revealed that indeed when IRS-1 levels
were examined in this set of patients, high IRS-1 expression was
associated with a shortened DFS. These data support the concept that
IRS-1 is an important molecule in ER-mediated growth, and that when
IRS-1 is coexpressed with ER, tumor recurrence is more frequent.
In summary, we provide evidence that IGFR1, IRS-1, and IRS-2 are
estrogen-regulated proteins. Increased expression of all of these
components leads to enhanced IGF signaling, resulting in synergistic
growth. Combined with the ability of IGF-I to transcriptionally
activate the ER (47, 48, 49, 50), this reveals complex cross-talk and synergism
between these important signal transduction pathways that results in
both pathways reinforcing each other. Further evidence for the
importance of IGF components in ER action and breast cancer growth is
provided by the prognostic significance of IRS-1 expression in
ER-positive breast cancer patients. Thus, this and other data provide
strong motivation for development of strategies to inhibit IGF action
in human breast cancer.
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MATERIALS AND METHODS
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Materials
All materials and chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted. IGF-I was
purchased from GroPep Pty. Ltd. (Adelaide, Australia). ICI
182780 was a kind gift from Zeneca Pharmaceuticals (Macclesfield,
U.K.). All tissue culture reagents were purchased from Life Technologies (Gaithersburg, MD) unless otherwise stated.
Cell Lines
MCF-7 cells have been maintained in our laboratory for many
years (51). ZR-75 and T47D human breast cancer cells were purchased
from the American Type Culture Collection (Manassas, VA).
MDA-MB-435A cells were kindly provided by Nils Brunner (Finsen
Laboratories, Copenhagen, Denmark). Cells were routinely maintained in
improved MEM (IMEM) + 10% fetal bovine serum (Summit Biotechnology, Ft. Collins, CO) + 2 mM
glutamine + 50 I.U./ml penicillin, 50 µg/ml streptomycin. SFM
consisted of IMEM + 10 mM HEPES pH 7.4, 1 µg/ml
transferrin, 1 µg/ml fibronectin, 2 mM glutamine, 50
I.U./ml penicillin, 50 µg/ml streptomycin, and trace elements
(Biofluids, Rockville, MD).
Cell Stimulation and Lysis
Cells were plated at 5 x 105 cells in 6-cm
dishes (Becton Dickinson and Co., Lincoln Park, NJ) and
allowed to adhere overnight. The next day the medium was changed to
SFM, and 24 h later cells were stimulated with various ligands for
the indicated times. The concentrations were: E2,
10-9 M; Tam, 10-6
M; ICI, 10-6 M; and IGF-I, 5
x 10-9 M. After stimulation cells were washed
twice with cold PBS and then lysed in 150 µl of TNESV buffer with
fresh protease inhibitors (50 mM Tris-HCl, pH 7.4, 1%
NP-40, 3 mM EDTA, 100 mM NaCl, 10
mM sodium orthovanadate, 1 mM
phenylmethylsulfonylfluoride, 20 µg/ml leupeptin, and 20
µg/ml aprotinin). Lysates were clarified by centrifugation at
14,000 x g for 15 min at 4 C, and lysates were stored
at -20 C. Protein concentrations were determined by the bichionic acid
method according to the manufacturers instructions (Pierce Chemical Co., Rockford, IL).
Immunoblotting and Immunoprecipitation
Total protein (50 µg) was resuspended in denaturing sample
loading buffer (3% dithiothreitol, 0.1 M Tris-HCl, pH 6.8,
4% SDS, 0.2% bromophenol blue, 20% glycerol), separated by 8%
SDS-PAGE, and electrophoretically transferred to nitrocellulose
overnight at 4 C. The membrane was blocked with 5% milk-TBST (0.15
M NaCl, 0.01 M Tris-HCl, pH 7.4, 0.05% Tween
20). For anti-PY immunoblot, the membrane was incubated with a
1000:1 dilution of hrp-linked primary antibody (RC20,
Transduction Laboratories, Inc., Lexington, KY) in TBST.
Bands were visualized by ECL according to the manufacturers
instructions (Pierce Chemical Co.). For activated MAPK
(New England Biolabs, Inc., Beverly, MA) immunoblot, the
membrane was incubated with a 1000:1 dilution of primary antibody in
TBST. All other antibodies were diluted in TBST + 5% milk and used at
a concentration of 1000:1 for IRS-1, IRS-2, and total MAPK
(Upstate Biotechnology, Inc., Lake Placid, NY) and 200:1
for IGFR1 (Santa Cruz Biotechnology, Inc., Santa Cruz,
CA).
RNAse Protection Assay
MCF-7 cells were plated at 3 x 106 cells in
15-cm dishes (Becton Dickinson and Co.) and allowed to
adhere overnight. Cells were harvested by trypsin/EDTA and pelleted in
15-cm tubes. Total RNA was prepared by Qiagen RNeasy Midi Kit
(Qiagen, Valencia, CA) according to the manufacturers
instructions and checked for integrity by separation on a 1% agarose
gel. Ribonuclease (RNAse) protection was performed according to our
previously published method (15), and RNA loading was normalized to
mRNA of the ribosomal protein 36B4 (52), which has previously been
shown to be not regulated by estrogen. The IRS-1 cDNA was generated by
PCR from MCF-7 genomic DNA using a 5'-primer containing an
XbaI restriction site (5'-AGTTTCTAGACTCCAGCCCTGTTTGCATGT-3')
and a 3'-primer with an EcoRI restriction site
(5'-CGAAGAATTCGTCAGCCCGCTTGTTGATGT-5'). The probe for IGFR1 (53) has
been detailed previously.
Analysis of Human Tumors
Measurement of IRS-1 expression in 200 node-negative breast
cancer patients and association with other clinical and laboratory
factors have been described previously (7). DFS was defined as the time
from date of diagnosis to the date of first recognition of relapse or
last contact (censored). ER-positive (
3 fmol/mg) tumors were
dichotomized into those with IRS-1 levels above or below median IRS-1
value (0.61 arbitrary units) of the entire sample. DFS curves were
estimated by the Kaplan-Meier method (54) and compared using the
log-rank test.
Nude Mouse Model System
MCF-7 cells were grown in nude mice as xenografts as previously
described in detail (55). Estrogen supplementation was provided in the
form of a 3-week release 0.25-mg E2 pellet
(Innovative Research of America, Rockville, MD).
When the tumors had reached 810 mm in size, the mice were randomized
into continued estrogen treatment or removal of the E2
pellet. After a further month, the mice were killed and the tumors were
removed and treated as above (see Immunoblotting and
Immunoprecipitation).
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. S. Oesterreich and G. Chamness for helpful
comments and suggestions.
 |
FOOTNOTES
|
---|
Address requests for reprints to: A. V. Lee, Ph.D., Department of Medicine, Division of Medical Oncology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284-7884. E-mail:
adrian{at}oncology.uthscsa.edu
This work was supported by Public Health Service Grants P01CA-30195 and
P50CA-5818306 and Cancer Center Support Grant P30CA-54174 from the
National Cancer Institute (NIH).
1 These authors contributed equally to the manuscript. 
Received for publication July 13, 1998.
Revision received January 19, 1999.
Accepted for publication February 3, 1999.
 |
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