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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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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 estrogen’s growth promoting effects.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). 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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). 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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), 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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). 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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),
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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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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, 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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 2–8 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 (24–48 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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 manufacturer’s 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 manufacturer’s
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 manufacturer’s
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 8–10 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).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by Public Health Service Grants P01CA-30195 and P50CA-58183–06 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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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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L-A Martin, S Pancholi, C M W Chan, I Farmer, C Kimberley, M Dowsett, and S R D Johnston The anti-oestrogen ICI 182,780, but not tamoxifen, inhibits the growth of MCF-7 breast cancer cells refractory to long-term oestrogen deprivation through down-regulation of oestrogen receptor and IGF signalling Endocr. Relat. Cancer, December 1, 2005; 12(4): 1017 - 1036. [Abstract] [Full Text] [PDF]
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 S. B. Choi, J. S. Jang, and S. Park Estrogen and Exercise May Enhance {beta}-Cell Function and Mass via Insulin Receptor Substrate 2 Induction in Ovariectomized Diabetic Rats Endocrinology, November 1, 2005; 146(11): 4786 - 4794. [Abstract] [Full Text] [PDF]
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 X. Cui, R. Schiff, G. Arpino, C. K. Osborne, and A. V. Lee Biology of Progesterone Receptor Loss in Breast Cancer and Its Implications for Endocrine Therapy J. Clin. Oncol., October 20, 2005; 23(30): 7721 - 7735. [Abstract] [Full Text] [PDF]
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D Montanaro, M Maggiolini, A G Recchia, R Sirianni, S Aquila, L Barzon, F Fallo, S Ando, and V Pezzi Antiestrogens upregulate estrogen receptor {beta} expression and inhibit adrenocortical H295R cell proliferation J. Mol. Endocrinol., October 1, 2005; 35(2): 245 - 256. [Abstract] [Full Text] [PDF]
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T Frogne, J S Jepsen, S S Larsen, C K Fog, B L Brockdorff, and A E Lykkesfeldt Antiestrogen-resistant human breast cancer cells require activated Protein Kinase B/Akt for growth Endocr. Relat. Cancer, September 1, 2005; 12(3): 599 - 614. [Abstract] [Full Text] [PDF]
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Y. Yamaguchi, H. Takei, K. Suemasu, Y. Kobayashi, M. Kurosumi, N. Harada, and S.-i. Hayashi Tumor-Stromal Interaction through the Estrogen-Signaling Pathway in Human Breast Cancer Cancer Res., June 1, 2005; 65(11): 4653 - 4662. [Abstract] [Full Text] [PDF]
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M Koda, M Sulkowska, L Kanczuga-Koda, and S Sulkowski Expression of insulin receptor substrate 1 in primary breast cancer and lymph node metastases J. Clin. Pathol., June 1, 2005; 58(6): 645 - 649. [Abstract] [Full Text] [PDF]
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W. Ruan, M. E. Monaco, and D. L. Kleinberg Progesterone Stimulates Mammary Gland Ductal Morphogenesis by Synergizing with and Enhancing Insulin-Like Growth Factor-I Action Endocrinology, March 1, 2005; 146(3): 1170 - 1178. [Abstract] [Full Text] [PDF]
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M. Karl, M. Potier, I. H. Schulman, A. Rivera, H. Werner, A. Fornoni, and S. J. Elliot Autocrine Activation of the Local Insulin-Like Growth Factor I System Is Up-Regulated by Estrogen Receptor (ER)-Independent Estrogen Actions and Accounts for Decreased ER Expression in Type 2 Diabetic Mesangial Cells Endocrinology, February 1, 2005; 146(2): 889 - 900. [Abstract] [Full Text] [PDF]
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J. A. Nagle, Z. Ma, M. A. Byrne, M. F. White, and L. M. Shaw Involvement of Insulin Receptor Substrate 2 in Mammary Tumor Metastasis Mol. Cell. Biol., November 15, 2004; 24(22): 9726 - 9735. [Abstract] [Full Text] [PDF]
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 R. I. Fernando and J. Wimalasena Estradiol Abrogates Apoptosis in Breast Cancer Cells through Inactivation of BAD: Ras-dependent Nongenomic Pathways Requiring Signaling through ERK and Akt Mol. Biol. Cell, July 1, 2004; 15(7): 3266 - 3284. [Abstract] [Full Text] [PDF]
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 V. Bourdeau, J. Deschenes, R. Metivier, Y. Nagai, D. Nguyen, N. Bretschneider, F. Gannon, J. H. White, and S. Mader Genome-Wide Identification of High-Affinity Estrogen Response Elements in Human and Mouse Mol. Endocrinol., June 1, 2004; 18(6): 1411 - 1427. [Abstract] [Full Text] [PDF]
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 W.-F. Chen and M.-S. Wong Genistein Enhances Insulin-Like Growth Factor Signaling Pathway in Human Breast Cancer (MCF-7) Cells J. Clin. Endocrinol. Metab., May 1, 2004; 89(5): 2351 - 2359. [Abstract] [Full Text] [PDF]
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 H. Yu, X.-O. Shu, B. D. L. Li, Q. Dai, Y.-T. Gao, F. Jin, and W. Zheng Joint Effect of Insulin-like Growth Factors and Sex Steroids on Breast Cancer Risk Cancer Epidemiol. Biomarkers Prev., October 1, 2003; 12(10): 1067 - 1073. [Abstract] [Full Text] [PDF]
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E Nardon, I Buda, G Stanta, E Buratti, M Fonda, and L Cattin Insulin-like growth factor system gene expression in women with type 2 diabetes and breast cancer J. Clin. Pathol., August 1, 2003; 56(8): 599 - 604. [Abstract] [Full Text] [PDF]
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S. R.D. Johnston, T. Hickish, P. Ellis, S. Houston, L. Kelland, M. Dowsett, J. Salter, B. Michiels, J. J. Perez-Ruixo, P. Palmer, et al. Phase II Study of the Efficacy and Tolerability of Two Dosing Regimens of the Farnesyl Transferase Inhibitor, R115777, in Advanced Breast Cancer J. Clin. Oncol., July 1, 2003; 21(13): 2492 - 2499. [Abstract] [Full Text] [PDF]
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A. V. Lee, P. Zhang, M. Ivanova, S. Bonnette, S. Oesterreich, J. M. Rosen, S. Grimm, R. C. Hovey, B. K. Vonderhaar, C. R. Kahn, et al. Developmental and Hormonal Signals Dramatically Alter the Localization and Abundance of Insulin Receptor Substrate Proteins in the Mammary Gland Endocrinology, June 1, 2003; 144(6): 2683 - 2694. [Abstract] [Full Text] [PDF]
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E. R. Levin Bidirectional Signaling between the Estrogen Receptor and the Epidermal Growth Factor Receptor Mol. Endocrinol., March 1, 2003; 17(3): 309 - 317. [Abstract] [Full Text] [PDF]
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 S. R. D. Johnston, J. Head, S. Pancholi, S. Detre, L.-A. Martin, I. E. Smith, and M. Dowsett Integration of Signal Transduction Inhibitors with Endocrine Therapy: An Approach to Overcoming Hormone Resistance in Breast Cancer Clin. Cancer Res., January 1, 2003; 9(1): 524s - 532s. [Abstract] [Full Text]
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G. Bompard, C. Puech, C. Prebois, F. Vignon, and G. Freiss Protein-tyrosine Phosphatase PTPL1/FAP-1 Triggers Apoptosis in Human Breast Cancer Cells J. Biol. Chem., November 27, 2002; 277(49): 47861 - 47869. [Abstract] [Full Text] [PDF]
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X. Zhang and D. Yee Insulin-like Growth Factor Binding Protein-1 (IGFBP-1) Inhibits Breast Cancer Cell Motility Cancer Res., August 1, 2002; 62(15): 4369 - 4375. [Abstract] [Full Text] [PDF]
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H. Varma and S. E. Conrad Antiestrogen ICI 182,780 Decreases Proliferation of Insulin-like Growth Factor I (IGF-I)-treated MCF-7 Cells without Inhibiting IGF-I Signaling Cancer Res., July 15, 2002; 62(14): 3985 - 3991. [Abstract] [Full Text] [PDF]
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 B. A. Stoll Oestrogen/insulin-like growth factor-I receptor interaction in early breast cancer: clinical implications Ann. Onc., February 20, 2002; 13(2): 191 - 196. [Abstract] [Full Text] [PDF]
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 G. Thordarson, A. V. Lee, M. McCarty, K. Van Horn, O. Chu, Y.-C. Chou, J. Yang, R. C. Guzman, S. Nandi, and F. Talamantes Growth and characterization of N-methyl-N-nitrosourea-induced mammary tumors in intact and ovariectomized rats Carcinogenesis, December 1, 2001; 22(12): 2039 - 2047. [Abstract] [Full Text] [PDF]
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R. L. Stephen, L. E. Shaw, C. Larsen, D. Corcoran, and P. D. Darbre Insulin-like Growth Factor Receptor Levels Are Regulated by Cell Density and by Long Term Estrogen Deprivation in MCF7 Human Breast Cancer Cells J. Biol. Chem., October 19, 2001; 276(43): 40080 - 40086. [Abstract] [Full Text] [PDF]
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 E. R. Levin Genome and Hormones: Gender Differences in Physiology: Invited Review: Cell localization, physiology, and nongenomic actions of estrogen receptors J Appl Physiol, October 1, 2001; 91(4): 1860 - 1867. [Abstract] [Full Text] [PDF]
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R. G. Richards, D. M. Klotz, M. R. Bush, D. K. Walmer, and R. P. DiAugustine E2-Induced Degradation of Uterine Insulin Receptor Substrate-2: Requirement for an IGF-I-Stimulated, Proteasome-Dependent Pathway Endocrinology, September 1, 2001; 142(9): 3842 - 3849. [Abstract] [Full Text] [PDF]
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L. M. Shaw Identification of Insulin Receptor Substrate 1 (IRS-1) and IRS-2 as Signaling Intermediates in the {alpha}6{beta}4 Integrin-Dependent Activation of Phosphoinositide 3-OH Kinase and Promotion of Invasion Mol. Cell. Biol., August 1, 2001; 21(15): 5082 - 5093. [Abstract] [Full Text] [PDF]
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S. Oesterreich, P. Zhang, R. L. Guler, X. Sun, E. M. Curran, W. V. Welshons, C. K. Osborne, and A. V. Lee Re-expression of Estrogen Receptor {alpha} in Estrogen Receptor {alpha}-negative MCF-7 Cells Restores both Estrogen and Insulin-like Growth Factor-mediated Signaling and Growth Cancer Res., August 1, 2001; 61(15): 5771 - 5777. [Abstract] [Full Text] [PDF]
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T. W. Chan, M. Pollak, and H. Huynh Inhibition of Insulin-like Growth Factor Signaling Pathways in Mammary Gland by Pure Antiestrogen ICI 182,780 Clin. Cancer Res., August 1, 2001; 7(8): 2545 - 2554. [Abstract] [Full Text] [PDF]
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 J Dupont and D Le Roith Insulin-like growth factor 1 and oestradiol promote cell proliferation of MCF-7 breast cancer cells: new insights into their synergistic effects Mol. Pathol., June 1, 2001; 54(3): 149 - 154. [Abstract] [Full Text]
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D. L. Hadsell, T. Alexeenko, Y. Klemintidis, D. Torres, and A. V. Lee Inability of Overexpressed des(1-3)Human Insulin-Like Growth Factor I (IGF-I) to Inhibit Forced Mammary Gland Involution Is Associated with Decreased Expression of IGF Signaling Molecules Endocrinology, April 1, 2001; 142(4): 1479 - 1488. [Abstract] [Full Text]
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 R. Clarke, F. Leonessa, J. N. Welch, and T. C. Skaar Cellular and Molecular Pharmacology of Antiestrogen Action and Resistance Pharmacol. Rev., March 1, 2001; 53(1): 25 - 72. [Abstract] [Full Text] [PDF]
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M. B. Martin, T. F. Franke, G. E. Stoica, P. Chambon, B. S. Katzenellenbogen, B. A. Stoica, M. S. McLemore, S. E. Olivo, and A. Stoica A Role for Akt in Mediating the Estrogenic Functions of Epidermal Growth Factor and Insulin-Like Growth Factor I Endocrinology, December 1, 2000; 141(12): 4503 - 4511. [Abstract] [Full Text] [PDF]
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C. K. Osborne and S. A. W. Fuqua Selective Estrogen Receptor Modulators: Structure, Function, and Clinical Use J. Clin. Oncol., September 17, 2000; 18(17): 3172 - 3186. [Abstract] [Full Text] [PDF]
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A. V. Lee, J. L. Gooch, S. Oesterreich, R. L. Guler, and D. Yee Insulin-Like Growth Factor I-Induced Degradation of Insulin Receptor Substrate 1 Is Mediated by the 26S Proteasome and Blocked by Phosphatidylinositol 3'-Kinase Inhibition Mol. Cell. Biol., March 1, 2000; 20(5): 1489 - 1496. [Abstract] [Full Text]
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J. G. Jackson, T. Yoneda, G. M. Clark, and D. Yee Elevated Levels of p66 Shc Are Found in Breast Cancer Cell Lines and Primary Tumors with High Metastatic Potential Clin. Cancer Res., March 1, 2000; 6(3): 1135 - 1139. [Abstract] [Full Text]
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G. Pete, C. R. Fuller, J. M. Oldham, D. R. Smith, A. J. D’Ercole, C. R. Kahn, and P. K. Lund Postnatal Growth Responses to Insulin-Like Growth Factor I in Insulin Receptor Substrate-1-Deficient Mice Endocrinology, December 1, 1999; 140(12): 5478 - 5487. [Abstract] [Full Text]
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J. Dupont, M. Karas, and D. LeRoith The Potentiation of Estrogen on Insulin-like Growth Factor I Action in MCF-7 Human Breast Cancer Cells Includes Cell Cycle Components J. Biol. Chem., November 10, 2000; 275(46): 35893 - 35901. [Abstract] [Full Text] [PDF]
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A. Lai, B. Sarcevic, O. W. J. Prall, and R. L. Sutherland Insulin/Insulin-like Growth Factor-I and Estrogen Cooperate to Stimulate Cyclin E-Cdk2 Activation and Cell Cycle Progression in MCF-7 Breast Cancer Cells through Differential Regulation of Cyclin E and p21WAF1/Cip1 J. Biol. Chem., July 6, 2001; 276(28): 25823 - 25833. [Abstract] [Full Text] [PDF]
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S. Kahlert, S. Nuedling, M. van Eickels, H. Vetter, R. Meyer, and C. Grohe Estrogen Receptor alpha Rapidly Activates the IGF-1 Receptor Pathway J. Biol. Chem., June 9, 2000; 275(24): 18447 - 18453. [Abstract] [Full Text] [PDF]
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