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Theses and Dissertations
2005
Regulation of estrogen receptor signaling in breast
and endometrial cancer by the src kinase pathway,
the micronutrient selenium, and by novel
tamoxifen-regulated biomarkers
Yatrik M. Shah
Medical College of Ohio
Follow this and additional works at: http://utdr.utoledo.edu/theses-dissertations
Recommended Citation
Shah, Yatrik M., "Regulation of estrogen receptor signaling in breast and endometrial cancer by the src kinase pathway, the
micronutrient selenium, and by novel tamoxifen-regulated biomarkers" (2005). Theses and Dissertations. 1459.
http://utdr.utoledo.edu/theses-dissertations/1459
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Regulation of Estrogen Receptor Signaling in Breast and
Endometrial Cancer by the Src Kinase Pathway, the
Micronutrient Selenium, and by Novel Tamoxifen-regulated
Biomarkers
Yatrik M. Shah
Medical College of Ohio
2005
ACKNOWLEDGEMENTS
I would like to thank my major advisor for his support and valuable instructions. I would
like to thank the members of my advisory committee, Drs. Manohar Ratnam, Sonia
Najjar, Kam Yeung, and Douglas Pittman for their advice and guidance throughout my
graduate school years. I would like to acknowledge my appreciation to our collaborators
Drs. Yan Dong and Clement Ip on the selenium projects. I like to acknowledge Dr.
Fernand Labrie for the generous gift of EM-652, and Dr. Robert Clarke for the MCF-7LCC2 cell line. I like to express thanks to all the members of Dr. Rowan’s lab for their
assistance and support.
ii
TABLE OF CONTENTS
Acknowledgements------------------------------------------------------------------------- ii
Table of Contents--------------------------------------------------------------------------- iii
Introduction---------------------------------------------------------------------------------- 1
Literature------------------------------------------------------------------------------------- 5
Manuscript 1 Title: Selective Estrogen Receptor Modulator Regulated
Proteins in Endometrial Cancer Cells. --------------------------------------------------- 33
Manuscript 2 Title: The Src Kinase Pathway Promotes Tamoxifen
Agonist Action in Ishikawa Endometrial Cells through PhosphorylationDependent Stabilization of Estrogen Receptor α Promoter
Interaction and Elevated SRC-1 Activity. -----------------------------------------------79
Manuscript 3 Title: Attenuation of Estrogen Receptor α (ERα)
Signaling by Selenium in Breast Cancer Cells via Downregulation
of ERα Gene Expression.------------------------------------------------------------------142
Manuscript 4 Title: Selenium Disrupts Estrogen Receptor α Signaling and
Restores Tamoxifen Sensitivity in Tamoxifen Resistant
Breast and Endometrial Cancer Cells. -----------------------------------------------------180
Summary-------------------------------------------------------------------------------------- 223
Bibliography---------------------------------------------------------------------------------- 226
Abstract---------------------------------------------------------------------------------------- 292
iii
INTRODUCTION
Estrogens are important in the growth and maintenance of female reproductive
tissues and other non-reproductive tissues such as bone, cardiovascular tissue, and the
central nervous system. Estrogen action is mediated through binding to its cognate
receptor, estrogen receptor (ER). Two forms of ER have been identified, ERα and ERβ,
both the product of separate genes (Kuiper et al., 1996; Mosselman et al., 1996; Tremblay
et al., 1997). The ER is a ligand-dependent transcription factor, and upon estrogen
binding the receptor either forms heterodimers or homodimers. Upon dimerization, ER
binds to the promoter region of target genes where subsequent recruitment of coactivators
and local chromatin modifications facilitate gene transcription (Evans, 1988; KleinHitpass et al., 1986). In addition ER is activated by ligand-independent mechanisms that
involve crosstalk from peptide and growth factor signal transduction pathways (Levin,
2003). In the absence of the physiological ligand 17β-estradiol, cellular kinases can
activate ER and modulate ER-regulated gene transcription.
The ER plays a major role in the development and growth of breast cancer. As a
treatment for breast cancer, the selective estrogen receptor modifier (SERM) tamoxifen
binds with high affinity to the ER and blocks estrogen induced cellular growth (Dutertre
et al., 2000). Tamoxifen is the most widely used hormonal therapy for breast cancer and
is approved as a chemopreventive agent in high risk women (Jordan, 2000). As an ER
antagonist, tamoxifen inhibits estrogen dependent gene transcription that is required for
cellular growth in breast tissue and breast-derived cell lines (Dutertre et al., 2000; Jordan,
2000; Shang et al., 2002). Despite the benefits of tamoxifen therapy, almost all
1
tamoxifen-responsive breast cancer patients develop resistance to therapy (Osborne,
1998). In addition, tamoxifen exhibits ER agonist action in the uterus that manifests in an
increase incidence of endometrial cancer (Fornander et al., 1989; Harper et al., 1967;
Terenius, 1971).
Several hypotheses are proposed to explain tamoxifen resistance and the estrogenlike activity exhibited in the endometrium. Coregulators are important in the mechanism
of tamoxifen resistance. Elevated levels of amplified in breast cancer 1 (AIB-1) (RAC3/ACTR/SRC-3) in patients receiving tamoxifen treatment are correlated with tamoxifen
resistance (Osborne et al., 2003). Elevated steroid receptor coactivator-1 (SRC-1) (NcoA1), but not glucocorticoid receptor interacting protein-1 (GRIP-1) (TIF-2/NcoA-2/ SRC2) or AIB-1 correlate with tamoxifen agonist activity in the Ishikawa endometrial cell
line (Shang et al., 2002). In addition to coactivators, there is a decrease in nuclear
corepressor (N-CoR) levels with the onset of tamoxifen resistance in both human breast
cancer samples and in mouse models of breast cancer (Girault et al., 2003; Lavinsky et
al., 1998). Also, 4-hydroxytamoxifen acts as a full agonist on ER-dependent transcription
in fibroblast cells isolated from the N-CoR null mouse. However, in wild-type mouse
embryo fibroblasts containing normal N-CoR levels, 4-hydroxytamoxifen did not activate
ER-dependent transcription (Jepsen et al., 2000).
In addition to coregulator protein expression, several deregulated cellular kinase
pathways play an important role in tamoxifen resistance and tamoxifen-mediated
endometrial proliferation. As mentioned above ligand-dependent or -independent
pathways can activate ER and both modes of ER activation are modulated by receptor
2
phosphorylation. Estrogen receptor is a phosphoprotein and the major phosphorylation
sites of ER reside in the N-terminal domain (NTD) at serines 104, 106, 118, and 167
(Arnold et al., 1994b; Kato et al., 1995; Le Goff et al., 1994). Overexpression of erb2/HER2 an epidermal growth factor receptor (EGFR) family member are associated with
tamoxifen resistance (Benz et al., 1993; Liu et al., 1995; Pietras et al., 1995). Erb2/HER2 are upstream of extracellular regulated kinase (ERK)-1 and ERK-2, which
phosphorylate ERα on serine 118 and enhance its transcriptional activity upon estrogen
and tamoxifen incubation (Kato et al., 1995). In addition, AKT kinase protects breast
cancer cells from tamoxifen-mediated apoptosis, and the consensus AKT site serine 167
on ERα is required (Campbell et al., 2001). MEKK1 increases the agonist activity of
tamoxifen in endometrial cells implicating downstream mitogen activated protein kinases
(MAPKs) in regulating tamoxifen agonist/antagonist action (Lee et al., 2000). Src
kinase, which activates both MAPK and AKT can enhance the transcriptional activity of
the tamoxifen in breast and cervical cancer cell lines (Feng et al., 2001).
Coregulator proteins are also substrates for cellular signaling pathways. SRC-1 is
phosphorylated on seven consensus sites for proline-directed protein kinases in vivo
(Rowan et al., 2000a;b). The GRIP-1 and AIB-1 are phosphorylated in vitro by MAP
kinases (Font et al., 2000; Lopez et al., 2001). In breast cancer cells the transcriptional
activity of AIB-1 was increased by MAPK phosphorylation that stimulated recruitment of
histone acetyltransferases (Font et al., 2000). Recently, it was shown that AIB-1 is
phosphorylated on six sites important for its full transcriptional activity (Wu et al., 2004).
Furthermore, cellular signaling cascades also phosphorylate corepressors. Interaction of
3
N-CoR and silencing mediator for retinoid and thyroid hormone (SMRT) with
progesterone receptor (PR) decreased after incubation with 8-bromo cAMP (Wagner et
al., 1998). In vitro phosphorylation by MEKK decreased the interaction of SMRT with
thyroid receptor (Hong et al., 1998).
Currently the mechanisms of tamoxifen resistance and tamoxifen agonist actions
in the endometrium are undefined. An increased use of tamoxifen for breast cancer
therapy and chemoprevention highlights an urgent need to 1) identify reliable biomarkers
that can predict with high certainty the response of breast cancer to tamoxifen therapy, 2)
understand the precise molecular mechanism which contributes to the development of
acquired tamoxifen resistance as well as estrogen-like agonist action in the endometrium
and, 3) develop non-cross-resistant therapies designed to deplete ERα and/or disrupt ER
signaling that can be used alone or in combination with tamoxifen to alleviate the
unwanted side-effects.
The studies described in this dissertation include: the identification of proteins
associated with the growth stimulatory effects of tamoxifen in the ERα-positive,
tamoxifen resistant Ishikawa endometrial adenocarcinoma cell line using twodimensional gel electrophoresis; examining the role of non-receptor tyrosine kinase src in
promoting the tamoxifen agonist action in the endometrium; assessing the role of the
micronutrient selenium in ER signaling and providing a mechanism-based rationale for
combining selenium with tamoxifen as a therapeutic strategy to improve the efficacy of
tamoxifen in both breast cancer treatment and chemoprevention.
4
LITERATURE
Nuclear Receptor Activation
Nuclear receptors (NR) are a superfamily of liganded transcription factors and are
important in a wide variety of cellular processes (Evans, 1988; Tsai et al., 1994). Nuclear
receptors can be classified into type 1 and type 2 receptors. Type 1 receptors consist of
PR, ER, androgen receptor (AR), glucocorticoid receptor (GR), and mineralocorticoid
receptor (MR). Upon specific ligand binding, type 1 receptors dimerize and binds to
specific elements termed response elements in the promoter of genes (Nelson et al.,
1999a). Subsequent recruitment of coactivator proteins initiates transcription. In contrast
type 2 receptors such as thyroid receptor (TR), all-trans retinoic acid receptor (RAR), 9cis retinoic acid receptor (RXR), and vitamin D3 receptor (VDR) are already bound to
DNA in their unliganded state bound with corepressor proteins that repress basal
transcription. Upon ligand binding the corepressor interaction is lost, coactivators are
recruited, and transcription is initiated.
Estrogen Receptor
Estrogen is a female sex hormone important in the female reproductive tract. The
main source of estrogen produced in the body is from the ovaries through a complex
series of hydroxylation, oxidation, and aromatization reactions that convert 21-carbon
cholesterol to 18-carbon estrogen. The production of ovarian estrogen is under control of
two hormones lutenizing hormone (LH) and follicle stimulating hormone (FSH). The LH
stimulates the production of androgens, whereas FSH activates aromatase enzymes that
5
convert androgens to estrogens. Once formed, estrogen exerts it effects on female
reproductive tissues. During the proliferative phase of the mentrual cycle when estrogen
concentration is the highest, the endometrium becomes 3 to 5 fold thicker and uterine
glands become enlarged. Estrogen stimulates proliferation and keritinization in the
vagina. During puberty when estrogen concentrations begin to rise, the mammary gland
becomes enlarged due to the increase in ductal size and accumulation of adipose tissue
surrounding the ducts (Ryan, 1999).
The specificity of estrogen in tissues is due to the presence of a type 1 NR, ER.
Two isoforms of ER have been identified, ERα and ERβ (Kuiper et al., 1996; Mosselman
et al., 1996; Tremblay et al., 1997). The ER is part of the nuclear receptor/ thyroid
superfamily and like all NR, ERα and ERβ contain six modular domains each with a
specific functional property (Kumar et al., 1987) (Figure 1). The N-terminal A/B domain
(NTD) contains a ligand-independent activation function 1 (AF-1) (Evans, 1988; Tora et
al., 1989; Tsai et al., 1994; Webb et al., 1998). A centrally located DNA binding domain
(DBD) that contains two zinc fingers motifs is important in binding DNA (Evans, 1988;
McKenna et al., 1999; Tsai et al., 1994). A hinge region contains a nuclear localization
sequence. The E/F domain or ligand binding domain (LBD) includes sites for hormone
binding, ligand dependent activation function 2 (AF-2), and contains the dimerization
domain (Evans, 1988; McKenna et al., 1999; Tsai et al., 1994). The ERα and ERβ are
84% to 97% homologous in the DBD, 54% homologous in the LBD but only 28%
homologous in the NTD (Enmark et al., 1997; Mosselman et al., 1996) (Figure 1).
Targeted deletion of ER in mice demonstrated that ERα was the predominant ER isoform
6
Figure 1. Schematic Diagram of ERα and ERβ
Structure Demonstrating Amino Acid Homology
Between Different ER Domains
AF-1
Domains
Amino
Acid Homology
ERα
ERβ
1
1
DNA
Ligand/AF-2
A/B
C
D
E
F
26%
84%
12%
58%
12%
180
263
302
552 593
144
227
255
504 530
7
in the mammary gland and the uterus. The uterus is comprised of three tissue
compartments (myometrium, endometrial stroma, luminal/glandular epithelium) and both
ERα and ERβ are expressed in all three compartments (Hiroi et al., 1999). ERα -/- mice
are hypoplastic in all three compartments and whole uterine wet weight is half that of
wild type mice whereas uteri of the ERβ -/- mice appear similar to wild type mice (Krege
et al., 1998). In the mammary gland, ERβ -/- mice do not exhibit abnormalities
compared to age matched wild type female mice (Krege et al., 1998). The ERα knockout
mice exhibit decreased ductal growth and pregnancy differentiation of the mammary
gland (Bocchinfuso et al., 1997). The presence of ERα and ERβ and estrogen synthesis in
the fetal ovaries suggests a role for both ERα and ERβ in ovarian development (Sar et al.,
1999; Weniger, 1990); however, no histological abnormalities were found with either
ERα -/- or ERβ -/- mice. At sexual maturity discrete ovarian abnormalities became
apparent. ERα -/- mice were not able to ovulate; whereas, ERβ -/- had considerably
lower litter size compared to wild type mice (Krege et al., 1998; Lubahn et al., 1993;
Schomberg et al., 1999). Surprisingly, targeted deletions of both ERα and ERβ displayed
a different phenotype than the individual ERα -/- or ERβ -/-, clearly demonstrating a role
for both ER isoforms in the ovaries (Couse et al., 1999). In addition to estrogen effects on
reproductive physiology, estrogens are important in non-classical estrogen target tissues.
Estrogens play an important role in brain function. Epidemiological studies demonstrate
a protective effect of hormone replacement therapy (HRT) on neurodegenerative diseases
such as Alzeiheimer’s disease (Wise et al., 2001). Estrogen-mediated protection of
8
neurons required ERα and not ERβ (Dubal et al., 2001), although the mechanisms are
undefined. Estrogens also exert effects on bone. It has been well characterized that an
increase in osteoporosis is correlated with a decrease of estrogen levels during
menopause (Watts, 1999). Both ERα and ERβ are expressed in localized areas of the
bone (Bord et al., 2001; Braidman et al., 2001; Vidal et al., 1999). Furthermore, both
ERα and ERβ are important in bone formation and remodeling (Jessop et al., 2004; Lee
et al., 2004). Estrogens are also important in the cardiovascular system (Bakir et al.,
2000; Dubey et al., 2000), adipose tissue (Homma et al., 2000), and in male reproduction
(Couse et al., 2001) via ERα and/or ERβ.
Nuclear Receptor Coactivators
Estrogen Receptor action is potentiated by a group of proteins termed
coactivators. Numerous NR coactivator proteins have been identified in recent years. A
coactivator is a protein that interacts with NR and potentiates nuclear receptor activity. A
coactivator interacts with the NR through an LXXLL motif (where L is leucine and X is
any amino acid) termed the NR box or receptor interaction domain (RID) (Heery et al.,
1997) (Figure 2). Chromatin structure is a key regulator of basal transcription. Histones
repress transcription through tight binding of the negatively charged DNA by positively
charged lysine side chains. By acetylation of lysine residues in histones, the interaction is
disrupted and the DNA is no longer tightly bound to histone proteins. When the
chromatin is in a relaxed conformation it is more conducive to transcription due to
accessibility of the transcription machinery. The well characterized p160 family of
9
Figure 2. General Schematic Diagram of p160
Coactivators and N-CoR and SMRT Corepressors.
P160 Coactivator
Q
S/T
PAS
PAS
bHLH
160 kDa
RID
RID
RID
Corepressor
250-300 kDa
RD1
RD2
RD3
RID
SANT
Domains
bHLH: helix loop helix region; PAS: Per=ARNT-Sim Domain; S/T:
Serine Threonine rich region; Q: Glutamine rich region; RID: Receptor
Interaction Domain; RD: Repressor Domain
10
coactivators include SRC-1 (Kamei et al., 1996; Onate et al., 1995), GRIP-1 (Hong et al.,
1996; Voegel et al., 1996), and AIB-1 (Anzick et al., 1997; Chen et al., 1997; Li et al.,
1997; Takeshita et al., 1996; Torchia et al., 1997). The p160 family of coactivators either
contain intrinsic histone acetyltransferase (HAT) activity or recruit other HATs such as
p300/CBP associated factor (P/CAF) and Creb Binding Protein (CBP) (Chakravarti et al.,
1996; Kamei et al., 1996; Spencer et al., 1997). The p160 family of coactivators and CBP
function synergistically in enhancing transcription (Chen et al., 1997; Liu et al., 2001;
Spencer et al., 1997). However, site-directed mutagenesis targeting the HAT motif on
p160 coactivators did not significantly affect its ability to coactivate nuclear receptors
(Liu et al., 2001; Spencer et al., 1997), suggesting that p160 family members may be
important in the assembly of other transcriptions factors and coactivators on the
promoter. Recent studies demonstrate a dynamic model of coactivator recruitment where
ER immediately binds to DNA followed by the sequential recruitment of p160
coactivators, P300, and the TR associated protein (TRAP)/VDR interacting protein
(DRIP) complex. Local chromatin structure is modified that facilitates RNA polymerase
II binding. Subsequently, CBP acetylation of p160 coactivators leads to coactivator and
ER release. Following ER release, CBP and pCAF disassemble and the cycle is repeated
(Burakov et al., 2002; Shang et al., 2000).
In addition to histone acetylases, recently identified coactivator-associated
arginine methyltransferase 1 (CARM1) and protein arginine N-methyltransferases
(PMRTs) provide yet another mechanism of chromatin remodeling. The CARM1 and
PRMT bind to the c-terminal region of SRC-1 and methylate histones H3 and H4. The
11
intrinsic methyltransferase property of these proteins is required for coactivator activity
(Chen et al., 1999; Wang et al., 2001b). In contrast the ubiquitin ligase activity of
coactivator E6-associated protein (E6-AP) is not required for coactivation of NR in vitro
and in vivo (Nawaz et al., 1999a; Sivaraman et al., 2000; Smith et al., 2002). Another
essential group of coactivators are represented by the TRAP/DRIP complex (Burakov et
al., 2000; Fondell et al., 1996; Rachez et al., 1998). The TRAP/DRIP complex acts to
stabilize the nuclear receptor:coactivator complex at the promoter and bridge the NR
complex to the basal transcriptional machinery (Burakov et al., 2000; Gu et al., 1999;
Kim et al., 1994).
The importance of each p160 family member on ER action has been demonstrated
in vivo with targeted deletions of SRC-1, GRIP-1, and AIB-1. SRC-1 -/- mice exhibit
normal growth and fertility; however, these mice exhibit decreased uterine growth
following exposure to estrogen. In addition, there is decreased mammary gland
formation and partial resistance to estrogen (Xu et al., 1998). Interestingly, in adult rats
an acute knockdown of SRC-1 in the brain results in an increase of GRIP-1 expression
(Apostolakis et al., 2002). Similar compensatory expression of GRIP1 was demonstrated
in sertoli cells (Mark et al., 2004). Therefore, the modest effects on reproductive
phenotypes in SRC-1-/- mice may be due to genetic compensation by GRIP-1. The
GRIP-1 -/- mice display a decrease in male and female fertility (Gehin et al., 2002). In
addition, GRIP-1 also affects thermogenesis and energy expenditure (Picard et al., 2002).
The AIB-1 -/- mice exhibit significant growth retardation and reduced body size. There is
a decrease in the estrogen levels of AIB-1 -/- mice that delay pubertal development and
12
mammary gland growth (Wang et al., 2000; Xu et al., 2000). In a mammary gland tumor
model, AIB-1 -/- mice exhibited reduced tumor formation compared to wild-type mice
(Kuang et al., 2004).
Nuclear Receptor Corepressor
In contrast to coactivators, corepressors are NR interacting proteins that repress
NR activity. Corepressors are associated with unliganded type II receptors or antagonist
bound type I receptors. Two well-studied corepressors are N-CoR and SMRT (Chen et
al., 1995; Horlein et al., 1995) (Figure 2). Similar to coactivators, corepressors contain
RID domains important in interaction with the NR. The RID domain, of corepressors
share similar homology to the coactivator-RID domain, and consist of LXXI/HIXXXI/L
(where L is leucine, I is isoleucine, histidine, and X is any amino acid). The SMRT and
N-CoR mediate transcriptional repression by recruiting histone deacetylase complexes
that remove acetyl groups from histone proteins, and thus confer a tight conformation
repressing basal transcription (Heinzel et al., 1997; Nagy et al., 1997). Targeted deletion
of N-CoR demonstrated the importance of transcriptional repression for mammalian
development. The N-CoR deficient embryos died at day 15.5. The N-CoR is needed for
active repression of several NR and other transcription factors. In addition, N-CoR was
required for the antagonist activity of antiestrogen compounds (Jepsen et al., 2000).
Classical Ligand-Dependent ER Activation
Estrogen is a lipophilic molecule that crosses the cell membrane and binds to the
LBD of the ER. Upon ligand binding the receptor dissociates from heat shock protein-90
(HSP-90) and other chaperone proteins. The receptor either forms heterodimers
13
consisting of ERα-ERβ or homodimers of two ERα or ERβ (Cowley et al., 1997; Ogawa
et al., 1998; Pettersson et al., 1997). Upon dimerization, ER binds to an estrogen response
element (ERE) 5′ GGTCAnnnTGACC 3′, a palidromic inverted repeat in the promoters of
estrogen dependent genes (Klein-Hitpass et al., 1986). The binding of an agonist to the
ER stabilizes helix 12 in the AF-2 region. The conformation allows coactivator proteins
to recognize an amphipathic α-helix on the surface of the agonist-bound LBD formed by
helix 3,4,5, and 12 via the NR box (Shang et al., 2000; Steinmetz et al., 2001;
Weatherman et al., 1999) (Figure 3).
In addition to activating transcription at an ERE, ERα and ERβ activate
transcription at AP1 sites (Figure 3). In contrast to the classical mechanism of ER
activation, ER regulation of transcription via AP1 sites does not involve direct binding to
DNA, but instead ER binds to jun/fos proteins that, in turn, bind to AP-1 consensus
sequences on DNA (Paech et al., 1997b; Webb et al., 1995, 1999). It is hypothesized that
ER binds to coactivator proteins recruited by jun/fos and this interaction increases the
transactivation capacity of the coactivators. The ER also activates transcription at ERE
half sites adjacent to SP1 sites. ER interacts with the c-terminal binding domain of SP1
proteins to transactivate genes (Porter et al., 1997). Additionally, it is found that ER/SP1
complexes activated transcription at GC rich elements independent of ER binding to
DNA, similar to what is observed in the ER/AP1 paradigm (Dong et al., 1999; Qin et al.,
1999; Samudio et al., 2001; Wang et al., 2001a; Xie et al., 1999). A transgenic mouse
model has been developed that does not allow ERα binding through the
14
Figure 3. Estrogen Receptor Activation
E2
EGF
mER
Nongenomic
Cellular
Response
Ligand-Dependent
Ligand-Independent
Kinases
HSP
HSP
ER
ER
P
ER ER
or
at
it v
c
oa
C
R
ERE
P ER
Jun
ER ER
Fos
Coactivator
ER
E
E
Coactivator
AP-1
ER is activated by four pathways. 1) Activation of ER at EREcontaining promoters by the classical ligand-dependent pathway. 2)
Activation of ER at ERE-containing promoters by ligand-independent
pathways. 3) Activation of ERE-independent promoters by either liganddependent or independent pathways. 4) Nongenomic signaling by a
membrane-associated binding site. E2: Estradiol; mER:Membrane ER
15
classical ERE-mediated mechanism but retains the ability of ERα to bind to AP1 and SP1
proteins. Mice heterozygous for the mutant ERα are infertile preventing generation of
homozygous mice. Although more studies are needed, the transgenic model provides
strong evidence for ERE-independent mechanisms in vivo. Interestingly, mice
heterozygous for ERα +/- are fertile suggesting that the mutant allele act in a dominant
negative manner to inhibit estrogen-regulated physiology (Jakacka et al., 2002).
Ligand-Independent/Crosstalk with signaling Pathways
In the absence of estrogen, mitogenic growth factors stimulate ERα
transcriptional activity (Figure 3). Both ligand-dependent and -independent ERα
activation is modulated by phosphorylation and site-specific phosphorylation of ERα
increases its transcriptional activity (Ali et al., 1993; Bunone et al., 1996; El Tanani et al.,
1997; Lahooti et al., 1994; Le Goff et al., 1994; Washburn et al., 1991; Weigel et al.,
1998). Major phosphorylation sites of ERα are in the NTD at serine 104 (Le Goff et al.,
1994), serine 106 (Le Goff et al., 1994), serine 118 (Kato et al., 1995), serine 167
(Arnold et al., 1994). In addition there is one site in the DBD at serine 236, one site in
the hinge region at serine 305 (Wang et al., 2002), and two sites in the LBD at threonine
311 (Lee et al., 2002) and tyrosine 537 (Arnold et al., 1997). Numerous signaling
pathways phosphorylate ER (REVIEWED IN Coleman et al., 2001; Lannigan, 2003).
Mutation of serines 104, 106, 118, and 167 to alanine results in a general decrease in
ERα transcriptional activity (Arnold et al., 1995a; Castano et al., 1997; Le Goff et al.,
1994; Tzeng et al., 1996). Serine 118 is phosphorylated by MAPK and is important for
coactivator binding (Bunone et al., 1996; Dutertre et al., 2003; Endoh et al., 1999; Kato et
16
al., 1995). Serine 167 is a consensus AKT site and phosphorylation mediates DNA
binding (Arnold et al., 1995a; Castano et al., 1997; Martin et al., 2000; Sun et al., 2001b;
Tzeng et al., 1996) and promoter interaction. Serine 305 is phosphorylated by protein
kinase A (PKA) and p21 activated kinase-1 (PAK1) and is important in the
transcriptional activity of ERα (Balasenthil et al., 2004; Michalides et al., 2004).
Threonine 311 is downstream of P38 kinase and is important for nuclear export of the
receptor (Lee et al., 2002). Tyrosine 537 is phosphorylated by src kinase in vitro (Arnold
et al., 1995b) and important in DNA binding (Arnold et al., 1995c). In addition specific
amino acid substitutions (Y537S and Y537A) have led to constitutive activation of ERα
(Weis et al., 1996).
There is a well-characterized reciprocal crosstalk between ERα and growth factor
signaling pathways. Numerous in vitro experiments provide evidence of crosstalk
between ER and growth factor signaling (REVIEWED IN Levin, 2003). The best in vivo
examples of crosstalk were demonstrated in the uterus. Estrogen and growth factors
stimulate proliferation in the endometrium (Nelson et al., 1991). In the insulin-like
growth factor-2 (IGF-1) knockout mouse, estrogen-induced uterotropic effects were
significantly retarded in spite of functional ERα present in the uterus (Adesanya et al.,
1999). Also in the ERα knockout mice, no uterotropic effects were detected upon
epidermal growth factor (EGF) treatment, even though EGF signaling was intact (Klotz
et al., 2002). The role of phosphorylation in ligand-independent crosstalk has been well
characterized for ERα. In contrast, phosphorylation and cellular signaling crosstalk is
not defined for ERβ; however, ERβ was activated via a ligand-independent pathway and
17
phosphorylated in the AF-1 region (Chu et al., 2004; Coleman et al., 2003; Tremblay et
al., 1999).
In addition to ER, coregulator proteins are targets of signaling pathways. SRC-1
contains seven in vivo phosphorylation sites and all seven sites contain a consensus
sequence for proline-directed protein kinases (Rowan et al., 2000b). In addition, the
induction of protein kinase A (PKA) enhanced ligand-independent activation of PR
through phosphorylation of SRC-1 (Rowan et al., 2000a). In in vitro studies both GRIP-1
and AIB-1 were phosphorylated by MAP kinase, ERK-2 (Font et al., 2000; Lopez et al.,
2001). In breast cancer cells the transcriptional activity of AIB-1 increased via MAPK
phosphorylation that stimulated the recruitment of other HATs (Font et al., 2000). The
AIB-1 contain six phosphorylation sites important for full transcriptional activation (Wu
et al., 2004). Also interaction of N-CoR and SMRT with PR decreased after incubation
with 8-bromo cAMP (Wagner et al., 1998) and in vitro phosphorylation by MEKK
decreased the interaction of SMRT with thyroid receptor (Hong et al., 1998).
Nongenomic Estrogen Signaling
Ligand-dependent and -independent ER-regulated actions are considered genomic
pathways due to the involvement of ER-mediated gene transcription. A distinct estrogenmediated pathway referred to as nongenomic signaling exists that does not require
mRNA and protein synthesis (Figure 3). A membrane receptor is thought to mediate
nongenomic estrogen signaling (Bression et al., 1986; Brubaker et al., 1994; Marquez et
al., 2001). However, it is controversial whether a separate membrane receptor that is
distinct from the classical ER exists (Filardo et al., 2000; Filardo et al., 2002), or a
18
receptor that contains homology to the classical ER stimulates the nongenomic estrogen
response (Kim et al., 1999; Norfleet et al., 2000). Nongenomic estrogen signaling rapidly
(seconds-minutes) activate several signaling pathways that have significant physiological
consequences (Filardo et al., 2000; Kahlert et al., 2000; Migliaccio et al., 1996; Sun et al.,
2001a). Estrogens rapidly activate the MAPK and AKT pathways that can induce a
proliferative (Castoria et al., 1999; Migliaccio et al., 1996, 2000) and an anti-apoptotic
response (Castoria et al., 2001). In addition, nongenomic estrogen signaling stimulated
nitric oxide release (Stefano et al., 2000a), increased intracellular calcium levels (Stefano
et al., 2000b) and enhanced prolactin secretion (Watson et al., 1999).
Breast Cancer
The mammary gland consist of 6 to 10 major ducts that are subdivided into
lobules that are considered the functional unit of the mammary gland. Breast carcinomas
are divided into noninvasive and invasive carcinomas. Noninvasive carcinomas can be
subdivided into two major histological subtypes; ductal carcinoma in situ (DCIS) or
lobular carcinoma in situ (LCIS). The DCIS or LCIS do not have the capacity to
infiltrate the basement membrane, and therefore do not produce distant metastases
(Swain, 1989a,b). However, untreated DCIS and LCIS have an increased risk for
development of invasive carcinomas (Mallon et al., 2000; Stallard et al., 2001). Similar
to carcinoma in situ, there are two major histological subtypes of invasive carcinomas,
ductal and lobular, although other less frequent types exist (Dixon et al., 1982).
Epidemiological studies have shown age, diet, and genetic predispostions are risk factors
for breast cancer (McPherson et al., 2000).
19
The best characterized risk factor for breast cancer is hormonal exposure. As
early as 1896, it was shown that oophorectomy was a successful treatment for
management of breast cancer (Leake, 1996). In addition, early menarche and late
menopause that increase the exposure of estrogen in a lifetime can increase the risk of
breast cancer (Kelsey et al., 1991). Also multiple pregnancies that interrupt the menstrual
cycle and decrease the exposure to estrogen are protective against breast cancer (Hulka et
al., 1994; Russo et al., 1982). Women who receive exogenous estrogen have an
increased breast cancer risk (1997; Colditz et al., 1995; Magnusson et al., 1999).
The ERα is expressed in normal breast epithelium, although these cells do not proliferate
upon estrogen treatment. It is thought that estrogen induces the secretion of mitogenic
growth factors that stimulate adjacent ER-negative cells to proliferate via a paracrinemediated mechanism (Cunha et al., 1997). In contrast, the majority of premalignant
breast cells express ERα and are sensitive to estrogen-mediated proliferation and
regression upon estrogen ablation (Clarke et al., 1993, 1997; Johnston et al., 1994; Jordan
et al., 1975). Several animal models demonstrate the importance of ERα in breast
tumorigenesis. Transgenic overexpression of Erb2/Her2 or WNT under control of the
mouse mammary tumor virus (MMTV) promoter increased the incidence of mammary
tumors (Cardiff et al., 1993). Introduction of the MMTV-erb2/Her2 or MMTV-WNT
transgene into an ERα null background significantly delayed tumor onset indicating that
ERα is important for mammary tumorigenesis (Bocchinfuso et al., 1999; Hewitt et al.,
2002).
20
In contrast to ERα, the role of ERβ in breast cancer remains undefined. The ERβ
is expressed in the majority of normal breast epithelial cells and is present as several
splice variants that differ in functional properties compared to the full-length ERβ (Speirs
et al., 2004). There is limited data on the role of ERβ in breast cancer, but as breast
cancers progress from a benign to a more malignant phenotype, the ERα/ERβ is
increased with a predominance of ERα expression in the more malignant tumors (Leygue
et al., 1998).
Selective Estrogen Receptor Modulators (SERM)
As a treatment for breast cancer SERMs bind with high affinity to ER and act in a
tissue specific manner. Tamoxifen is a prototypic SERM and the most widely used
hormonal therapy for breast cancer (Jordan, 2000) (Figure 4). Tamoxifen therapy
profoundly improved the overall survival of breast cancer patients ( Breast Cancer
Trialists' Collaborative Group, 1998) without the adverse side-effects associated with
cytotoxic chemotherapy (Pritchard et al., 1997). Furthermore, tamoxifen is the only agent
approved for breast cancer chemoprevention in high risk women (Jordan, 2000). The
major mechanism of action for tamoxifen in breast cancer is as an ER antagonist.
Tamoxifen is a competitive inhibitor of estrogen and upon tamoxifen binding, ER recruits
a corepressor complex that inhibits estrogen dependent gene transcription (Dutertre et al.,
2000; Jordan, 2000; Shang et al., 2002). Tamoxifen is an estrogen antagonist in the
breast, but tamoxifen displays uterotropic effects in the endometrium (Harper et al., 1967;
Terenius, 1971). Tamoxifen therapy results in a 4-fold increase in the incidence of
endometrial cancer in postmenopausal women
21
Figure 4. Chemical Structure of SERMs and
Aromatase Inhibitors.
Estradiol
EM-652
Tamoxifen
Anastrozole
22
Raloxifene
Fulvestrant
(Fornander et al., 1989). New generation SERMs are being sought with the same or better
efficacy than tamoxifen but with fewer side effects.
Raloxifene, a SERM structurally distinct from tamoxifen, is approved for the
treatment and prevention of osteoporosis in postmenopausal women (Figure 4). In
addition, raloxifene does not display estrogen-like activity in the endometrium (Boss et
al., 1997; Delmas et al., 1997). Although, only modest effects of raloxifene were found in
ER-positive breast cancers (Gradishar et al., 2000), an on-going clinical trial is assessing
the effects of raloxifene on breast cancer incidence as a beneficial side effect to its antiosteoporosis activity (Cummings et al., 1999). Acolbifene (EM-652) is also in clinical
trial for breast cancer (Figure 4). EM-652 is a potent antiestrogen and does not display
estrogen-like action in mouse mammary gland and endometrium. Similar to raloxifene,
EM-652 preserves bone density in ovariectomized rats. In addition, EM-652 has
beneficial effects on plasma lipid profiles (Labrie et al., 2003) making EM-652 a
potentially attractive alternative to tamoxifen therapy. The search for SERMs with an
ideal tissue profile has been offset by the development of antiestrogenic agents that
inhibit ER action in all tissues. Fulvestrant (faslodex, ICI 182,780) is an ER antagonist
with no known agonist effects (Wakeling et al., 1991) (Figure 4). Fulvestrant binds to ER
and promotes ER protein degradation (McClelland et al., 1996). Fulvestrant is approved
for ER-positive metastatic breast cancer in postmenopausal women with disease
progression after tamoxifen therapy (Bross et al., 2003). Aromatase inhibitors have been
shown to be effective in inhibiting ER-positive breast cancers. Aromatase inhibitors
specifically target the rate-limiting enzyme needed for estrogen production. The
23
aromatase inhibitor agent anastrozole was more effective than tamoxifen as an adjuvant
endocrine treatment for ER-positive breast cancers (Figure 4). However, anastrozole was
also associated with an increase in bone fractures in women receiving compared to
patients receiving tamoxifen therapy (Howell et al., 2005).
Mechanisms of Tissue Specific SERM Effects
Currently there are several proposed mechanisms that explain the tissue specific
effects of SERMs. The different complement of coregulator proteins in the uterus vs. the
breast has been suggested to underlie tamoxifen agonist activity in the uterus. An
increase in corepressors levels decreased the agonist effect of tamoxifen. Whereas, an
increase in coactivator levels promoted the agonist activity of tamoxifen (Smith et al.,
1997). Shang et al. (2002) have demonstrated a specific increase of SRC-1 expression in
tamoxifen resistant Ishikawa endometrial cancer cells compared to tamoxifen sensitive
MCF-7 breast cancer cells. Ishikawa cells activated transcription of ER-dependent genes
following incubation with estrogen and tamoxifen via recruitment of SRC-1. Tamoxifen
treatment repressed transcription in MCF-7 cells via recruitment of corepressors (Shang
et al., 2002). It has been hypothesized that expression of AF-1 interacting coactivators
may regulate tissue-specificity of SERMs. Synergism between the AF-1 and AF-2 is
required for full activity of ERα; however, AF-1 and AF-2 can activate genes to a certain
extent independently (Kraus et al., 1995). Tamoxifen inhibits AF-2 but AF-1, a cell and
promoter specific activation function, is not blocked (Berry et al., 1990; Tzukerman et al.,
1994). It is suggested that if a cell contains a large number of AF-1 specific coactivators
24
such as p68 helicase and steroid receptor RNA activator (SRA) (Endoh et al., 1999; Lanz
et al., 1999), the tamoxifen-ER complex in this context may activate transcription.
Another variable to consider in the tissue specific activity of SERMs is the
ERα/ERβ expression ratios. The ERα and ERβ act in opposite regulatory mode on ERregulated AP1 elements upon estrogen and tamoxifen treatment (Paech et al., 1997a).
Moreover, ERβ is less transcriptionally active than ERα due to a decrease or absence of
AF-1 activity (Delaunay et al., 2000). In addition, ERβ can reduce the transcriptional
activity of ERα through hetreodimerization and/or competitive binding to ER-regulated
promoters (Hall et al., 1999). Therefore, tissue specific differences in the ERα/ERβ ratio
may potentially alter the pharmological response and modulate the tissue specific activity
SERMs.
Differentially activated cellular signaling pathways may regulate tissue specificity
of SERMs. As mentioned above, several signaling pathways target ER and coregulators
and these pathways can alter the relative antagonist/agonist property of tamoxifen.
Activation of PKA in tamoxifen sensitive MCF-7 breast cancer cells increased the
transcriptional activity of tamoxifen to levels similar to that of estrogen (Fujimoto et al.,
1994). Estrogen phosphorylation at serine 118 enhanced the transcriptional activation in
response to estrogen and tamoxifen (Kato et al., 1995). MEKK1 increased the agonistic
activity of tamoxifen implicating kinases downstream to be important in the cell
specificity of tamoxifen (Lee et al., 2000). Feng et al (2001) demonstrated that the
Src/JNK pathway enhanced the transcriptional activity of the tamoxifen-ER complex
through the AF-1 domain of the ER. It is doubtful that a single mechanism modulates the
25
tissue specific effect of SERMs. Rather, it is likely to be multifactorial encompassing
several mechanisms described above.
Tamoxifen Resistance
Clincial tamoxifen resistance is defined as the inability of tamoxifen to inhibit the
growth of hormone dependent breast tissue. Thirty to fifty percent of patients with ERαpositive breast cancer are resistant to tamoxifen therapy (ERα-positive de novo
resistance) (Osborne, 1998) and those patients that initially respond will eventually
become resistant to tamoxifen therapy (acquired resistance) (Osborne, 1998). The
majority of breast tumors that acquire tamoxifen resistance are ERα-positive (Johnston et
al., 1995) and are sensitive to second line hormonal therapy (Howell et al., 1995;
Osborne, 1998). Therefore, loss of ERα is not a major mechanism for acquired tamoxifen
resistance. Several ERα mRNA variants exist and many of these variant mRNAs are
translated in breast tumors (Murphy et al., 1998). Moreover, there is cell-based evidence
that tamoxifen can act as an agonist on specific ERα mutants (Jiang et al., 1992);
however, ERα mutations occur at very low frequency in tamoxifen resistant breast
tumors (Karnik et al., 1994). Thus it is unlikely that ERα mutations constitute a major
mechanism for tamoxifen resistance.
Other mechanisms that modulate ERα function may be important in tamoxifen
resistance. Overexpression of AIB-1 is a good prognostic marker of tamoxifen
resistance. Samples of 316 frozen breast tumors were assessed for AIB-1 expression. It
was found that patients receiving tamoxifen treatment who had elevated AIB-1 levels had
a decrease in disease free survival (Osborne et al., 2003). In contrast, low corepressor
26
levels were associated with tamoxifen resistance. A decrease in N-CoR levels correlated
with a shorter relapse free survival in patients undergoing tamoxifen therapy suggesting
that N-CoR may be a good independent prognostic marker of tamoxifen resistance
(Girault et al., 2003). In addition, a decrease in N-CoR levels correlated with the onset of
tamoxifen resistance in a mouse model for human breast cancer (Lavinsky et al., 1998).
Finally, 4-hydroxytamoxifen was a full agonist for ER-dependent transcription in
fibroblasts isolated from the N-CoR null mice compared to its antagonist activity in
fibroblasts from wild type mice (Jepsen et al., 2000).
Several ERβ variants and mutations have been characterized, but the role of ERβ
in tamoxifen resistance remains unknown (Reviewed in Speirs et al., 2004). A few
reports have shown a decrease in ERβ levels in tamoxifen resistant breast cancers
(Esslimani-Sahla et al., 2004; Hopp et al., 2004); however, more studies will be needed to
confirm the role of ERβ in tamoxifen resistance.
In addition to ER and coregulator expression, several deregulated cellular kinase
pathways were involved in tamoxifen resistance. Overexpression of erb-2/HER2 was
associated with tamoxifen resistance (Benz et al., 1993; Liu et al., 1995; Pietras et al.,
1995). The HER2 receptor is a member of the EFGR (HER1) family of transmembrane
receptor tyrosine kinases that also includes HER3 and HER4. HER2 is recruited as the
preferred partner of EGFR, HER3 or HER4 into an active, phosphorylated, heterodimeric
complex that activates several signaling pathways involved in the proliferation and
enhanced survival of tumor cells (Olayioye et al., 2000; Yarden et al., 2001)
27
Increased activity of MAPK and PI3K/AKT pathways was associated with
tamoxifen resistance. As mentioned earlier, both MAPK and PI3K/AKT pathways
phosphorylate and increase the transcriptional activity of ERα. Several studies
demonstrated a deregulation of MAPK activity following long-term tamoxifen treatment
(Berstein et al., 2003; Donovan et al., 2001; Rabenoelina et al., 2002). Furthermore,
breast cancer samples from patients undergoing anti-hormonal therapies that display an
increased MAPK activity were associated with decreased survival (Gee et al., 2001). In
addition, several studies demonstrated that constitutively active AKT confered tamoxifen
resistance in breast cells (Campbell et al., 2001; Clark et al., 2002). Similar to the tissue
specific effects of SERMs, it is likely that several mechanisms contribute to ERα-positive
de novo and acquired tamoxifen resistance.
Therapeutic Role of Signal Transduction Inhibitors (STI)
In addition to SERMs, new therapies for cancer utilizing inhibitors of signaling
pathways are emerging and several agent have been approved for cancer therapy or are
currently in clinical trial (Agus et al., 2005; Ardizzoni et al., 2004; Drevs, 2003; Druker
et al, 2001a, b; Elsayed et al., 2001; Graham et al., 2004; O'brien et al., 2004; Repp et al.,
2003; Slamon et al., 2001; Sridhar et al., 2003a, b; Traxler et al., 2001; Vogel et al.,
2002) (Table I). Numerous monoclonal antibodies targeting the extracellular domain of
EGFR have been developed (Crombet-Ramos et al., 2002; Overholser et al., 2000;
Wallace et al., 2000; Yang et al., 2001). As an alternative approach to antibody inhibitors
of cell surface receptors, cell permeable small molecule inhibitor of EGFR such as
ZD1839 (Iressa) are currently in phase III
28
Table I Signal Tranduction Inhibitors
Approved or in Clincal Trials.
Drug
Tyrosine Kinase Target
Company
Monoclonal
Antibodies
Herceptin
MDX-H210
2C4
C225
MDX-447
HER2/Neu (Vogel C.L. et al., 2002)
HER2/Neu (Repp R. et al., 2003)
HER2/Neu (Agus D.B. et al., 2003)
EGFR (ErbB-1) (Graham J. et al., 2004)
EGFR (ErbB-1) (Sridhar S.S. et al., 2003a)
Genentech
Mederex
Genentech
Imclone
Mederex
Signal Transduction
Inhibitor (STI)
STI-571(Gleevec)
ZD1839 (Iressa)
OSI-774
PKI-166
PTK-787
SU5416
SU6668
Novartis
AstraZeneca
OSI/Genentech
Novartis
Novatis/Schering
Sugen/Pharmacia
Sugen/Pharmacia
29
Abl, c-kit, PDGFR (O’brien S. et al., 2004)
EGFR (ErbB-1) (Elsayed Y.A. et al., 2001)
EGFR (ErbB-1) (Ardizzoni A. et al., 2004)
EGFR (ErbB-1); HER2 (Traxler P. et al., 2001)
VEGFR (Drevs J. 2003)
VEGFR (Sridhar S.S. et al., 2003b)
VEGFR (Sridhar S.S. et al., 2003b)
clinical trials for non-small cell lung carcinomas (NSCLC) and are promising candidates
for breast cancer (Elsayed et al., 2001). Herceptin, an approved agent currently used for
HER2 over-expressing breast cancer has been shown to be effective as monotherpy for
advanced breast cancer (Vogel et al., 2002). Combination of herceptin with several
standard chemotherapeutic agents resulted in an ever greater tumor regression and
survival (Slamon et al., 2001). Clinical trials are currently assessing combination therapy
of STIs with anti-hormonal agents (Reviewed in Ellis, 2004).
Selenium in Cancer Therapy and Chemoprevention
Selenium is a trace and an essential micronutrient shown to have beneficial effects
in cancer chemoprevention. Organic selenium agents, methylselenocysteine (MSC) and
seleno-L-methionine (Se-Met) used in clinical chemoprevention studies are metabolized
in tissues to the active selenium metabolite, methylselenol (Clark et al., 1996; Ip et al.,
1999; Wang et al., 2002) (Figure 5). Methylseleninic acid (MSA), developed for
assessing organic selenium effects in in vitro cell line experiments, is directly converted
to the active metabolite, methylselenol, via a non-enzymatic reaction (Ip et al., 2000a)
(Figure 5). Organic selenium compounds inhibit cellular proliferation and induce
apoptosis via modulation of cell cycle regulatory proteins and activation of several
caspases as well as reduction in bcl-2 (Dong et al., 2002a, b, 2003; Ip et al., 2000b, 2001;
Jiang et al., 2001, 2002; Lu et al., 1995; Sinha et al., 1996, 1997,1 999, 2001; Wang et al.,
2001c, 2002b; Zhu et al., 2002, 2003). A recent study demonstrated inhibition of AR
signaling by MSA that decreases the growth of prostate cancer cell lines (Dong et al.,
2004). An additional benefit of organic selenium compounds for chemoprevention is low
30
or absent toxicity (Clark et al., 1996; Ip et al., 2000a; Marshall, 2001; Nelson et al.,
1999b). In contrast, inorganic selenium compounds such as selenite are genotoxic and no
longer used for selenium chemoprevention (Ip et al., 1994; Medina et al., 2001; Patterson
et al., 1997; Sinha et al., 1996).
In a landmark clinical trial Clark et al. demonstrated the chemopreventative
effects of Se-Met against prostate, lung and colon cancer (Clark et al., 1996; DuffieldLillico et al., 2002). The protective effects of selenium have been demonstrated on
several other cancers such as breast and colorectal carcinomas (El Bayoumy et al., 2004;
Patrick, 2004). Selenium use has been limited to the chemopreventative setting.
Recently, a synergistic interaction of organic selenium compounds with the
topoisomerase 1 inhibitor, irinotecan on in vivo xenograft tumors was demonstrated (Cao
et al., 2004). This study provides a strong rationale to utilize selenium as a novel therapy
for overt cancer through combination with well-established chemotherapeutic and
hormonal agents.
31
Figure 5. Selenium Activation
Organic Selenium
Inorganic Selenium
Selenocysteine
GS-Se-SG
Reduction
Reactions
lyase
Hydrogen
Selenide
(H2Se)
GS-SeH
Methylselenol
(CH3SeH)
Methylseleninic
Acid
Metabolic pathways of organic selenium compounds to its active
metabolite methylselenol in cancer chemoprevention.
32
Selective Estrogen Receptor Modulator Regulated Proteins in Endometrial Cancer Cells
Yatrik M. Shah1, Venkatesha Basrur2, Brian G. Rowan1,3
Department of Biochemistry & Molecular Biology1, Department of Microbiology and
Immunology2 and Program in Bioinformatics & Proteomics/Genomics2,
Medical College of Ohio, Toledo, OH
3
Corresponding author:
Brian G. Rowan, Ph.D.
Department of Biochemistry & Molecular Biology
Medical College of Ohio
3035 Arlington Ave.
Toledo, OH 43614-5804
Tel#: 419-383-4134
Fax#: 419-383-6228
Email: [email protected]
33
Abstract
Tamoxifen is the primary hormonal therapy for breast cancer and is also used as a
breast cancer chemopreventative agent. A major problem with tamoxifen therapy is
undesirable endometrial proliferation. To identify proteins associated with the growth
stimulatory effects of tamoxifen in an ER-positive model, the present study profiled total
cellular and secreted proteins regulated by estradiol and selective estrogen receptor
modifiers (SERMs) in the Ishikawa endometrial adenocarcinoma cell-line using two-8
-7
dimensional gel electrophoresis. Following 24-hour incubation with 10 M estradiol, 10
-7
M 4-hydroxytamoxifen, or 10
M EM-652 (Acolbifene), nine proteins exhibited
significant increase in expression. The proteins identified were heat shock protein 90-α,
and -β, heterogeneous nuclear ribonucleoprotein F, RNA polymerase II-mediating
protein, cytoskeletal keratin 8, cytoskeletal keratin 18, ubiquitin-conjugating enzyme E218kDa and nucleoside diphosphate kinase B. These protein profiles may serve as novel
indices of SERM response and may also provide insight into novel mechanisms of
SERM-mediated growth.
34
Introduction
Estrogens are vital regulators of growth and maintenance of female reproductive
physiology and other non-reproductive tissues such as bone, cardiovascular tissue, and
the central nervous system (Mueller and Korach, 2001). The specificity for estrogen
action in tissues requires the expression of the type 1 nuclear receptor, estrogen receptor
(ER) (Evans, 1988; Tsai and O'Malley, 1994). Two forms of ER have been identified,
ER-α and ER-β, both the product of separate genes (Kuiper et al., 1996; Mosselman et
al., 1996; Tremblay et al., 1997). Similar to all nuclear receptors, ER-α and ER-β are
comprised of modular domains, each with its own functional properties (Kumar et al.,
1987). The N-terminal A/B domain (NTD) contains a ligand-independent activation
function 1 (AF-1) (Evans, 1988; Tora et al., 1989; Tsai and O'Malley, 1994; Webb et al.,
1998). A centrally located DNA binding domain (DBD), contains two zinc fingers
important for DNA binding (McKenna et al., 1999; Tsai and O'Malley, 1994). The hinge
region, contains a nuclear localization sequence and the E/F domain (LBD) includes sites
for hormone binding and ligand-dependent activation function 2 (AF-2) (McKenna et al.,
1999; Tsai and O'Malley, 1994; Webb et al., 1998). ER is a ligand-dependent
transcription factor, and upon estrogen binding the receptor either forms heterodimers
consisting of ER-α -ER-β (Cowley et al., 1997; Ogawa et al., 1998; Pettersson et al.,
1997) or homodimers of two ER-α or ER-β proteins. Upon dimerization, ER binds to the
promoter region of target genes where subsequent recruitment of coactivators and local
chromatin modifications facilitate gene transcription (Klinge, 2000; Klinge, 2001;
McKenna et al., 1999; McKenna and O'Malley, 2000).
35
Estrogens play a major role in the development and growth of breast cancer and
antiestrogen therapies have been devised to oppose estrogen action. Estrogen promotes
cell proliferation that increases the chance for random genetic errors to be permanently
introduced in the genome of somatic cells. As a treatment for breast cancer, the selective
estrogen receptor modifier (SERM) tamoxifen binds with high affinity to the ER and
blocks estrogen induced cellular growth (Dutertre and Smith, 2000; Jordan and Koerner,
1975). Tamoxifen is the most widely used hormonal therapy for breast cancer and has
been approved as a chemopreventive agent in high risk women (Jordan, 2000). The major
mechanism of action for tamoxifen is as an ER antagonist, although receptor-independent
mechanisms have been proposed (Mandlekar and Kong, 2001). As an ER antagonist,
tamoxifen inhibits estrogen dependent gene transcription that is required for cellular
growth in breast tissue and breast-derived cell lines.
Although tamoxifen is an estrogen antagonist in the breast, tamoxifen displays
estrogen-like uterotropic effects in the endometrium (Harper and Walpole, 1967;
Terenius, 1971). Tamoxifen therapy results in increased stromal thickening and
endometrial hyperplasia and long-term tamoxifen treatment is associated with a 4 fold
increase in endometrial cancer (Fisher et al., 1994; Fornander et al., 1989).
Several hypotheses have been proposed to explain the tissue specific actions of
tamoxifen and other SERMs. Differential coregulator expression between tissues may
account for SERM specificity. In experimental models, elevated corepressors facilitates
the antagonist action of tamoxifen whereas an increase in coactivators promotes the
tamoxifen agonist activity (Smith et al., 1997). It is also hypothesized that expression of
36
AF-1 interacting coactivators may drive the tissue-specificity of SERMs. Tamoxifen
inhibits AF-2 (Berry et al., 1990), but AF-1 is not blocked suggesting that tamoxifen may
display estrogen agonist action in tissues expressing AF-1 specific coactivators such as
p68 helicase (Endoh et al., 1999) and steroid receptor RNA activator (SRA) (Lanz et al.,
1999). Finally, differentially activated cellular signaling pathways in the uterus vs. the
breast may contribute to SERM specificity. (Feng et al., 2001; Kato et al., 1995; Lee et
al., 2000).
The problems with tamoxifen therapy have led to the development of new
generation SERMs, such as EM-652, that were developed to limit the estrogen-like action
of tamoxifen in the uterus. EM-652, the active metabolite of EM-800, does not display
estrogen-like action in mouse mammary gland and endometrium (Labrie et al., 1999;
Labrie et al., 2001; Luo et al., 1998; Martel et al., 1998; Sourla et al., 1997). In
experimental cell culture models, EM-652 inhibits the growth of human breast and
endometrial cancer cell lines (Couillard et al., 1998; Labrie et al., 1999; Labrie et al.,
2001; Simard et al., 1997). However, unlike other pure antiestrogens, EM-652 preserves
bone density in ovariectomized rats (Martel et al., 2000) making EM-652 a potentially
attractive alternative to tamoxifen therapy.
Coinciding with the widespread use of tamoxifen therapy and the development of
novel SERMs, relatively few SERM-regulated proteins have been identified. We set out
to identify novel biomarker proteins that are either direct mediators or surrogate markers
of the estrogen agonist action of tamoxifen and the antagonist action of EM-652 in
Ishikawa endometrial cancer cells. By focusing on proteins that are regulated by SERMs
37
of varying estrogenicity using two-dimensional protein electrophoresis and mass
spectrometry, we sought to identify proteins that may participate in novel pathways of
tamoxifen sensitivity and resistance. Furthermore, these proteins may have predictive
value for tamoxifen therapy and may themselves serve as novel tumor targets for therapy
and tumor image analysis. By using this approach, we eliminated the problem of
discordance between mRNA and protein inherent with the gene array approach.
38
Material and Methods
MTT Assay to measure cell proliferation
Ishikawa cells were plated in 24 well plates (6000 cells/well) and cultured in
phenol red free DMEM (Gibco, Grand Island, NY) containing 2% fetal bovine serum that
was charcoal stripped to remove endogenous steroids. 24 h after plating, the cells were
incubated with vehicle, estradiol (10-8 M) (Sigma, St. Louis, MO), 4-hydroxytamoxifen
(10-7 M and 10-10 M) (Sigma) or EM-652 (10-7 M and 10-10 M). The medium, estrogen and
antiestrogens were replaced every two days for a total of 9 days incubation. After 9 days,
the medium was replaced with 0.5mL of MTT reagent (ICN, Aurora, OH) (1mg/ml of
2,5-diphenyl tetrazolium bromide in PBS). After 1 h, the MTT reagent was removed and
200 µL of DMSO (Fisher Biotech, Fairlawn, NJ) was added to each well. The plates were
read at a wavelength of 595nm.
Luciferase assay to measure gene transcription.
Ishikawa cells were plated in 6-well plates as described above. The cells were
transfected with 500 ng of EREe1b-luciferase reporter using Fugene transfection reagent
(Roche, Madison, WI). 24 h post transfection, the cells were incubated with vehicle,
estradiol (10-8 M), 4-hydroxytamoxifen (10-7 M) or EM-652 (10-7 M) for 24 h. The cells
were scraped from the plates and centrifuged at 500 x g for 4 min and the resulting cell
pellet was lysed with luciferase cell lysis buffer (Promega, Indianapolis, IN). Following
centrifugation at 20,000 x g for 5 min to remove cellular debris, the supernatant was used
for standard luciferase assay measured using a Lumat luminometer (Perkin Elmer,
39
Downers Grove, IL). The luciferase values were normalized to protein measured by
Bradford assay (BioRad, Hercules, CA).
RNA extraction and cDNA synthesis.
Ishikawa cells were plated in 100 mm plates (2x106 cell/plate) in DMEM medium
as described above. The cells were incubated with vehicle, estradiol (10-8 M), 4hydroxytamoxifen (10-7 M) or EM-652 (10-7 M) for 2 hours. Cells were pelleted by
centrifugation at 500 x g for 4 min and total RNA was extracted using Trizol
(Invitrogen). Briefly, 300 µL of Trizol was added to the cell pellets and vortexed for 1
min. After incubation at room temperature for 10 min, 100 µL of 100% chloroform was
added and the solution was vortexed for 15 seconds. The solution was incubated at room
temperature for 3 min and then centrifuged for 10 min at 20,000 x g. The top aqueous
layer was removed and 600 µL of 100% isopropanol was added. After vortexing, the
solution was incubated at room temperature for 10 min followed by centrifugation at
20,000 x g for 10 min. The supernatant was removed and the RNA pellet was washed
with 300 µL of 70% ethanol in DEPC water. The pellet was resuspended in DEPC water
and the RNA concentration was quantified using a DU 640 spectrophotometer (Beckman
Coulter, Palo Alto, CA). RNA was diluted to 52 ng/µL and reverse transcribed using
TaqMan Kit reagents (Applied Biosystems, Foster City, CA). Briefly, 1 µL 10X Buffer,
2.2 µL MgCl2, 2 µL dNTPs, 0.5 µL random hexamers, 0.2 µL RNase inhibitor, and 0.25
µL reverse transcriptase were added to 200 ng of diluted RNA for a final volume of 10
µL. Reverse transcription was performed using a PTC-0150 MiniCycler (MJ Research,
Waltham, MA) using conditions described in the TaqMan Kit.
40
Real time RT-PCR.
Primers and probes were designed using Primer Express Version 2.0 (Applied
Biosystems). All primers and probes, except GAPDH, were manufactured by Integrated
DNA Technologies, Inc. (Coralville, IA) Primers and probes for GAPDH were designed
and manufactured by Applied Biosystems. The probes were dual-labeled with the
fluorescent dye 56-FAM at the 5’ end and 3BHQ-1 at the 3’ end.
Progesterone Receptor (PR):
FWD-5’-CTATGCAGGACATGACAACACAAA-3’
REV-5’-TGCCTCTCGCCTAGTTGATTAAG-3’
Probe-5’-56FAM CCTGACACCTCCAGTTCTTTGCTGACAAG/3BHQ-1/-3’
Lactoferrin (LF) :
FWD-5’-GACGAGCAGGGTGAGATTAAGTG-3’
REV-5’-ACCGGAAAGCCCCAGTGTA-3’
Probe-5’-/56FAM/TGCCCAACAGCAACGAGAGATACTACGG/3BHQ-1/-3’
Fwd primer, rev primer and probe for PR, LF, or GAPDH and Taqman Universal Master
Mix (Applied Biosystems) were added to 200ng of cDNA. Real-time RT-PCR was
performed using a GeneAmp 5700 Sequence Detection System (Perkin Elmer). PCR
conditions were as follows: samples were heated at 50° C for 2 min, then 95° C for 10
min, followed by 40 cycles of 95° C for 15 sec and 60° C for 1 min. Values were
quantified using the Comparative CT method. All samples were normalized to GAPDH
and calibrated to vehicle treated samples.
41
Two-dimensional gel electrophoresis
Ishikawa cells were plated in 150 mm plates (5 x 106 cells/plate) in DMEM
containing 2% charcoal stripped serum for 48 h. After incubation the media was replaced
with DMEM containing no serum and the cells were incubated with vehicle, estradiol
(10-8 M), 4-hydroxytamoxifen (10-7 M) or EM-652 (10-7 M) for an additional 24 h. For the
secreted proteins, the serum-free DMEM conditioned medium was removed following
the 24 h incubation with estrogen and antiestrogens. The medium was dialyzed against
25mM ammonium bicarbonate, and then lyophilized. The lyophilized proteins were
resuspended in 2D buffer (8 M urea, 2% chaps, 2% 3-10 non-linear carrier ampholytes,
60 mM DTT and a trace of bromophenol blue). To prepare proteins for the total cellular
fraction, the medium was removed and the cells were washed with 1X PBS. Cells were
scraped from the plates and centrifuged at 500 x g for 4 min. The resulting cell pellet was
resuspended in 2D buffer and the cell extract was centrifuged at 100,000 x g for 30 min
to remove cellular debris. Following protein determination by the Bradford assay, equal
amounts of protein from each drug incubation were used to rehydrate the 3-10 non-linear
immobilized pH gradient (IPG) strips overnight. The IPG strips were electrophoresed in
the first dimension using a gradient program increasing to 3500V, for a total of 19.5 h
using the Multiphor II Isoelectric Focusing System (Amersham, Uppsala, Sweden).
Following first dimension electrophoresis, the IPG strips were equilibrated for 10 min in
SDS PAGE equilibration buffer (50 mM tris-HCL, 6 M urea, 30% glycerol, 2% SDS, 1%
DTT, and a trace of bromophenol blue). The equilibrated IPG strips were electrophoresed
42
in the 2nd dimension on a 12% SDS PAGE gel for 14 h at 10 mA per gel. Proteins were
visualized by either silver-stain or Colloidal Coomassie blue stain (Invitrogen).
Protein identification by mass spectrometry.
From the 2D gels, protein spots that exhibited ligand-induced change in expression from
at least three separate experiments were excised using a clean surgical steel blade. The
gel piece was diced into 1 mm cubes and rehydrated in 0.1 M ammonium bicarbonate
buffer containing 0.4 µg sequencing grade-modified trypsin at 37°C for 14 h. The tryptic
peptides were extracted from the gel slices using 60% acetonitrile, 0.1% triflouroacetic
acid for 30 minutes at 30°C. The peptides were pooled and concentrated in a vacufuge to
about 20 µl volume. Two µls of the digest was separated on a reverse phase column
(Aquasil C18, 15 µm tip x 75 µm id x 5 cm Picofrit column, New Objectives, Woburn,
MA) using acetonitrile/1% acetic acid gradient system (5-75% acetonitrile over 35 min
followed by 95% acetonitrile wash for 5 min) at a flow rate of 250nl/min). The eluent
(peptides) was directly introduced into an ion-trap mass spectrometer (LCQ-Deca XP
Plus, ThermoFinnigan) equipped with a nano-spray source. The mass spectrometer was
set for analyzing the positive ions and operated on double play mode in which the
instrument was set to acquire a full MS scan and a collision induced dissociation (CID)
spectrum on the most abundant ion from the full MS scan (relative collision energy
~30%). Dynamic exclusion was set to collect two CID spectra on the most abundant ion
and then exclude it for 2 min. An initial database search of CID spectra was carried out
against an indexed human database using TurboSEQUEST (BioworksBrowser v 3.0,
43
ThermoFinnigan). Peptide CIDs which showed Xcorr and ∆Cn (delta Cn) of >2 and >0.2
respectively were considered positive and manually verified. Any un-interpreted CID
spectra were searched using the MS-Tag provision of Protein Prospector
(http://prospector.ucsf.edu), with default search settings, against human/mouse proteins in
the non-redundant and/or SwissProt databases.
Western Blot Analysis.
Ishikawa cells were incubated with agents and protein extract was prepared as
described above. 50 µg of each protein extract was separated by electrophoresis on an
SDS-PAGE gel. The gel was transferred to nitrocellulose membrane, and blocked in 5%
nonfat milk in tris-buffered saline/Tween 20 (TBST) (10 mM tris, pH 8, 150 mM NaCl,
0.1% Tween-20) for 1h at room temperature, followed by washes with 1x TBST. The
membranes were incubated with primary antibodies in 5% dried milk/TBST at 4º C for
16 hours. Primary antibodies used were cofilin non-muscle isoform, cytokeratin 8/18,
hnRNP F, UBCh7, NDPK B (Santa Cruz Biotechnology INC, Santa Cruz, CA) and
HSP90 (Upstate Charlottesville, VA). All antibodies were diluted 1:1000 in 5% dried
milk/TBST except cofilin and UBCh7, which were diluted 1:150 and 1:500, respectively.
Following primary antibody incubation, the membranes were washed with TBST and
incubated for 1 hour at room temperature with secondary biotinylated anti-rabbit, goat, or
mouse at 1:2000 depending on the primary antibody used. Following TBST washes, the
membranes were incubated with horseradish peroxidase streptavidin (1 µg/ml) for 1 hour
at room temperature and washed with TBST. The signal was developed with addition of
44
enhanced chemiluminescence (ECL) solution (1:1 mixture of solution A+B (A: 9.9 ml of
0.1 M Tris pH 8.5 + 100 µl luminol + 44 µl P-coumaric, B: 10 ml of 0.1 M tris pH 8.5 +
6 µl hydrogen peroxide). The membranes were exposed to Kodak XOMAT film, and the
signal was quantitated using a Kodak Digital Science 1D image analysis software station
440 system.
Data Analysis
All data are expressed as ± S.D. P values were calculated using one-way anova
Dunnett’s T Test. P<.05 was considered significant.
45
Results
Estrogen receptor-regulated gene expression and cell growth in Ishikawa cells.
The Ishikawa cell line is a well-characterized, endometrial adenocarcinoma cell
line in which tamoxifen exhibits similar agonist properties as estrogen. To validate earlier
published observations on tamoxifen and estrogen effects in Ishikawa cells and to
investigate the role of the SERM EM-652, Ishikawa cells were incubated with estradiol,
4-hydroxytamoxifen, EM-652 and ER-target gene expression and cell proliferation was
assessed. Estradiol and 4-hydroxytamoxifen, activated the ERE2e1b-lucferase reporter
but EM-652 antagonized the reporter in transfected cells (Fig.1 panel A). Similar results
were observed using real-time RT PCR to measure endogenous ER-dependent gene
expression, estradiol and 4-hydroxytamoxifen, but not EM-652, induced the lactoferrin
and progesterone receptor genes in non-transfected cells (Fig. 1 panel B). When cell
proliferation was assessed, estradiol and 4-hydroxytamoxifen, but not EM-652, induced
Ishikawa cell proliferation (Fig. 1, panel C). These results confirm the relative agonist
and antagonist actions of estradiol, 4-hydroxytamoxifen and EM-652 in the Ishikawa cell
line providing an adequate model to correlate the relative agonist activity of these
SERMs with the profile of proteins that are regulated following treatment with these
agents.
Identification of proteins regulated by estrogen and SERMs in Ishikawa cells.
46
A two-dimensional electrophoresis/mass spectrometry proteomic approach was
used to identify changes in protein expression following incubation with estradiol and
SERMs in Ishikawa cells. All drug treatments were performed in duplicates and
experiments were repeated at least three times. Only those protein spots that exhibited
consistent change in expression over all experiments were excised and sequence
identified by mass spectrometry. Inconsistently regulated protein spots were excluded
from further analysis. Several proteins in the whole cell extract were down regulated
following incubation of cells with estradiol and SERMs. However, there were
inconsistencies in the magnitude of reduced expression over several independent
experiments and these proteins were not pursued for further analysis. For this reason,
only proteins that were induced by estradiol and SERMs were sequence identified since
these proteins exhibited the greatest change in expression.
500 µg of whole cell lysate or 100 µg of proteins secreted into serum-free medium
(Figs. 2 and 4, respectively) were separated by two-dimensional gel electrophoresis
followed by visualization with colloidal Coomassie or silver staining. Visual analysis of
the gels identified fourteen proteins that were differentially regulated by estradiol and
SERMs in whole cell lysate and the secreted fraction. LC-Tandem-MS was used to
sequence identify eight regulated proteins from whole cell lysate and one protein from
the secreted fraction (data summarized in Table 1). In the whole cell lysate, three proteins
were induced by both estradiol and 4-hydroxytamoxifen (Keratin type I cytoskeletal 18
(ck18), Heterogeneous nuclear ribonucleoprotein F (hnRNP F), and Keratin type II
cytoskeletal 8 (ck 8)) (Fig. 3 panel B and C). Three proteins were induced by EM-652
47
alone: ubiquitin-conjugating enzyme E2-18kDa (UbcH7), RNA polymerase II subunit 5mediating protein (RMP) and cofilin, non-muscle isoform (Fig. 3 panel D). Two proteins
were induced by estrogen and EM-652: heat shock protein 90-α (HSP90- α) and heat
shock protein 90-β (HSP90- β) (Fig. 3 panel A).
Based on molecular weight and
isoelectric point (pI) only the N-terminal region of RMP was identified possibly because
of degradation. The c-terminal region of RMP was not detected possibly because of an
acidic pI that was outside the pH range of the isoelectric focussing step.
In the secreted fraction, a total of six proteins were observed to be differentially
regulated by estradiol and SERMs. Five proteins were induced by estradiol and 4hydroxytamoxifen and one protein was downregulated by EM-652 alone. Only one
protein could be identified because of the low protein present in the secreted fraction
(required silver staining to visualize) and poor recovery of tryptic peptides due to silver
stain. Several attempts were made at mass spectrometric identification, but only one of
the protein spots was identified as nucleoside diphosphate kinase B (NDKB). NDKB was
induced by estradiol and 4-hydroxytamoxifen (Fig. 4 panel E).
Confirmation of estrogen and SERM regulated proteins by Western blot analysis.
Western blot analysis and enhanced chemiluminescence quantitation was used to
confirm the proteins identified by mass spectrometry sequencing. Ishikawa cells were
incubated with estradiol and SERMs for 24 h. The cell extract and secreted proteins were
prepared as described above. All drug treatments were performed in duplicates and
experiments were repeated at least three times. The Western blot results were consistent
48
with the two-dimensional electrophoresis/mass spectrometry proteomic approach. In the
whole cell lysate Ck8, Ck18, and hnRNP F were induced by estradiol and 4hydroxytaomxifen (Fig 5 panel A and B). HSP90 was induced by estradiol and EM652
treatment (Fig 5 panel A) and UBCh7 and cofilin were induced by EM652 alone (Fig 5
panel A and B). In the secreted fraction NDPK B was induced by estradiol and 4hydroxytamoxifen (Fig 5 panel C). NDPK B was also induced in the whole cell lysate by
estradiol and 4-hydroxytamoxifen (Fig 5 panel A and B.). RMP expression could not be
confirmed by Western blot analysis because antibody to this protein is not currently
available. Because of the increased sensitivity of Western blot analysis compared to
Coomassie stained gels, the expression of Ck8, Ck18, HSP90 and cofilin was observed in
all treatment conditions. (Fig 5 panel A). Regardless, the relative induction of these
proteins by estradiol and SERMs as measured by Western blot analysis was identical to
that observed with Coomassie stained gels confirming the regulation of these proteins.
49
Discussion
Tamoxifen-induced growth of the endometrium is a major clinical problem for
women undergoing tamoxifen therapy for breast cancer. We sought to identify proteins
that were regulated by estrogen, tamoxifen (partial agonist), and EM-652 (antagonist) in
the Ishikawa endometrial adenocarcinoma cell line. Using two-dimensional proteomic
technology, nine estradiol- and SERM- regulated proteins were identified from whole cell
lysate and the secreted fraction of Ishikawa cells. Regulation of eight of these proteins
were confirmed by Western blot analysis (antibody to RMP was not available for
Western blot analysis). Among the identified proteins were chaperones proteins, RNA
and RNA polymerase binding proteins, cytoskeletal proteins, a ubiquitin ligase and a
multi-functional kinase. With the exception of HSP90, Ck 8, and Ck 18, all of the
proteins are novel estradiol and/or SERM regulated proteins. HSP90 has been shown to
be induced by estrogen in the endometrium and in several uterine-derived cell lines
(Ramachandran et al., 1988; Shyamala et al., 1989; Tang et al., 1995; Wu et al., 1996).
Although Ck 8 and Ck 18 were shown to be regulated by estrogen and tamoxifen in a
breast cancer cell line (Coutts et al., 1996), this is the first study to show estrogen and
tamoxifen regulation of Ck 8 and Ck 18 in an endometrial-derived cell line.
HSP 90 α and β are chaperone proteins important in the proper folding of
numerous proteins and important in nuclear receptor signaling (for review see ref. (Pratt
and Toft, 1997; Walter and Buchner, 2002)). The present finding of estradiol-dependent
induction of HSP 90 α and β in Ishikawa cells is consistent with the findings of several
other reports providing an internal control for estrogenic response (Ramachandran et al.,
50
1988; Shyamala et al., 1989; Tang et al., 1995; Wu et al., 1996). Remarkably, we also
found induction of HSP-90 α and β with EM-652, an ER antagonist in Ishikawa cells.
This is the first indication that ER ligands that exhibit opposite agonist and antagonist
properties in the same cell line can induce expression of the same chaperone protein. The
function of the estrogenic regulation on HSP90 is still unknown but it may serve as a
negative feedback to down regulate estrogen signaling in the endometrium. In addition,
induction of HSP90 by EM-652 could attenuate estrogen signaling in the endometrium.
Further studies will explore the function for HSP90 induction by these two ligands.
Estradiol and 4-hydroxytamoxifen, unlike EM-652, exhibit agonist properties in
Ishikawa cells and three proteins (Ck 8, Ck 18 and hnRNP F) were induced with these
agonist ER ligands. Cytokeratins are structural proteins involved in cellular organization
(for review see ref. (Kirfel et al., 2003)). There is also evidence that cytokeratins are
important in modulating external stimuli and signal cascades (for review see ref.
(Paramio and Jorcano, 2002)). The related protein, cytokeratin 19 (Ck 19), is regulated by
estrogen in breast cancer cell lines (Choi et al., 2000). Ck 8 and Ck 18 were shown to be
regulated by estrogen in T47D cells, a breast cancer cell line in which tamoxifen is an ER
antagonist unlike its action in Ishikawa cells. When T47D cells were grown in estrogendepleted medium, expression of Ck 8 and Ck 18 was lost but expression was restored
when exogenous estrogen was added to the medium. Interestingly, incubation of T47D
cells with tamoxifen caused a reduction of Ck 8 and Ck 18 protein expression (Coutts et
al., 1996). Taken together with the results of the present study in Ishikawa cells, Ck 8 and
51
Ck 18 could serve as prospective biomarkers for the mixed agonist/antagonist action of
tamoxifen.
hnRNP F was also induced by both estradiol and 4-hydroxytamoxifen in Ishikawa
cells. Over twenty members of the hnRNP family have been identified. These proteins
are important in gene expression, pre-mRNA splicing, silencing, packaging and transport
(Carson et al., 1998; Grabowski, 1998; Mayeda and Krainer, 1992; Nakielny and
Dreyfuss, 1997; Ostareck et al., 1997). hnRNP proteins contain RNA binding motifs with
different substrate binding specificities (Matunis et al., 1993). hnRNP F binds with high
affinity to polyG sequences (Matunis et al., 1994). At this time, a role for hnRNP F in ER
signaling has not been identified, although other hnRNP family members are regulated by
estrogen and may bind to other nuclear receptors (Arao et al., 2002; Powers et al., 1998).
Recently, Auboeuf et al. found that steroid receptors mediate RNA processing from
steroid response elements (Auboeuf et al., 2002), suggesting that nuclear receptors may
recruit hnRNP-containing protein complexes involved in both transcription and RNA
processing.
Three proteins (UbcH7, RMP, and Cofilin, non-muscle isoform) were induced
only with the antagonist EM-652. Our finding of induction of UbcH7 is the first report of
a protein involved in the ubiquitin pathway that is specifically regulated by a SERM.
UbcH7 is an E2-type ubiquitin carrier protein involved in the ubiquitin-mediated
proteolytic pathway (for review see ref. (Weissman, 2001)). Several proteins involved in
the ubiquitin-mediated proteolytic pathway have also been shown to be important in
estrogen signaling (Lonard et al., 2000; Nawaz et al., 1999a). A functional disruption of
52
the ubiquitin ligase E6-associated protein (E6-AP) in mice reduced estrogen mediated
growth of the uterus (Smith et al., 2002). UbcH7 is a functional binding partner of E6-AP
(Nuber et al., 1996) and is also important in the degradation of E6-AP and transcriptional
intermediary factor-2 (TIF-2), a nuclear receptor coactivator (Yan et al., 2003). These
findings taken together with results from the present study suggest that UbcH7 may be an
important mediator of estrogen and SERM signaling in endometrial cells.
RMP was induced only with EM-652 although the result could not be confirmed
by Western blot analysis. Interestingly, RMP binds to subunit 5 of RNA polymerase and
inhibits RNA polymerase II function (Dorjsuren et al., 1998). It is possible that induction
of RMP by EM-652 could participate in the antagonism of ER dependent gene expression
by EM-652. Cofilin was also selectively induced by EM-652. Cofilin is a multifunctional
protein important in actin depolymerization and in cell locomotion (Chen et al., 2001;
Maciver and Weeds, 1994; Nagaoka et al., 1995). The increase of cofilin expression may
be followed by changes in actin organization required for EM652-mediated repression of
cell growth.
In the secreted fraction of Ishikawa cells, NDPK B was induced by estradiol and
4-hydroxytamoxifen. Previous studies have identified NDPK B as a secreted protein
(Chopra et al., 2003; Donaldson et al., 2002; Willems et al., 2002) although it is possible
that induction of the cytoplasmic levels (Fig 5 panel C.) led to increased extracellular
shedding. NDPK B is encoded by the NM23-h2 gene. Currently, eight separate genes
have been cloned which belong to the NM23 family. NDPK is involved in the transfer of
phosphates from NTPs to NDPs (Walinder, 1968). Several studies have confirmed the
53
metastatic-suppressing property of NDPK A and NDPK B (Baba et al., 1995; Leone et
al., 1991; Leone et al., 1993; Nakamori et al., 1993; Steeg et al., 1988). In addition,
NDPK B has also been shown to increase cell proliferation (Caligo et al., 1995; Caligo et
al., 1996). The NDPK A gene is directly regulated by estrogen (Lin et al., 2002) but the
present study is the first report to find the NDPK B protein induced by both estrogen and
tamoxifen. Since estrogen and tamoxifen increase cell proliferation in Ishikawa cells, it
will be interesting to assess whether NDPK B participates in estrogen- and tamoxifendependent proliferation in Ishikawa and other endometrium-derived cell lines. Future
studies may determine whether NDPK B could serve as a biomarker for the tamoxifen
resistant phenotype. Since NDPK B is a secreted protein it could easily be assayed by
non-invasive procedures.
In summary, using two-dimensional proteomic technology we have identified
nine estradiol and SERM regulated proteins from the whole cell lysate and secreted
fraction in the Ishikawa cell line. It is likely that other proteins are regulated by estrogen
and SERMs but are not detected by the proteomic approach. A disadvantage of this
approach is visualization and identification of low abundance proteins. However, a
proteomic-based approach allows direct analysis of gene expression at the protein level.
By correlating the estradiol and SERM-induced proteins to effects of these ligands on
growth stimulation and gene expression in Ishikawa cells, we may identify proteins
involved in novel mechanisms of SERM action. These proteins may further be useful as
biomarkers for the development of SERM resistance. Further studies will be performed to
54
elucidate the roles of these proteins in estrogen and/or SERM-regulated gene expression
and cellular growth.
Acknowledgements: We are grateful to Dr. Fernand Labrie for the generous gift of EM652. This work was supported by a Predoctoral Dissertation Award from the Susan G.
Komen Breast Cancer Foundation (Y.K.S.) and a grant from the Cancer Research
Foundation of America (B.G.R.).
55
References
Arao, Y., Kikuchi, A., Ikeda, K., Nomoto, S., Horiguchi, H., and Kayama, F., 2002.
A+U-rich-element
RNA-binding
factor
1/heterogeneous
nuclear
ribonucleoprotein D gene expression is regulated by oestrogen in the rat uterus.
Biochem.J. 361, 125 132
Auboeuf, D., Honig, A., Berget, S. M., and O'Malley, B. W., 2002. Coordinate regulation
of transcription and splicing by steroid receptor coregulators. Science 298, 416
419
Baba, H., Urano, T., Okada, K., Furukawa, K., Nakayama, E., Tanaka, H., Iwasaki, K.,
and Shiku, H., 1995. Two isotypes of murine nm23/nucleoside diphosphate
kinase, nm23-M1 and nm23-M2, are involved in metastatic suppression of a
murine melanoma line. Cancer Res. 55, 1977 1981
Berry, M., Metzger, D., and Chambon, P., 1990. Role of the two activating domains of
the oestrogen receptor in the cell-type and promoter-context dependent agonistic
activity of the anti-oestrogen 4-hydroxytamoxifen. The EMBO Journal 9, 2811
2818
Caligo, M. A., Cipollini, G., Fiore, L., Calvo, S., Basolo, F., Collecchi, P., Ciardiello, F.,
Pepe, S., Petrini, M., and Bevilacqua, G., 1995. NM23 gene expression correlates
with cell growth rate and S-phase. Int.J.Cancer 60, 837 842
56
Caligo, M. A., Cipollini, G., Petrini, M., Valentini, P., and Bevilacqua, G., 1996. Down
regulation of NM23.H1, NM23.H2 and c-myc genes during differentiation
induced by 1,25 dihydroxyvitamin D3. Leuk.Res. 20, 161 167
Carson, J. H., Kwon, S., and Barbarese, E., 1998. RNA trafficking in myelinating cells.
Curr.Opin.Neurobiol. 8, 607 612
Chen, J., Godt, D., Gunsalus, K., Kiss, I., Goldberg, M., and Laski, F. A., 2001.
Cofilin/ADF is required for cell motility during Drosophila ovary development
and oogenesis. Nat.Cell Biol. 3, 204 209
Choi, I., Gudas, L. J., and Katzenellenbogen, B. S., 2000. Regulation of keratin 19 gene
expression by estrogen in human breast cancer cells and identification of the
estrogen responsive gene region. Mol.Cell Endocrinol. 164, 225 237
Chopra, P., Singh, A., Koul, A., Ramachandran, S., Drlica, K., Tyagi, A. K., and Singh,
Y., 2003. Cytotoxic activity of nucleoside diphosphate kinase secreted from
Mycobacterium tuberculosis. Eur.J.Biochem. 270, 625 634
Couillard, S., Gutman, M., Labrie, C., Belanger, A., Candas, B., and Labrie, F., 1998.
Comparison of the effects of the antiestrogens EM-800 and tamoxifen on the
growth of human breast ZR-75-1 cancer xenografts in nude mice. Cancer Res. 58,
60 64
57
Coutts, A. S., Davie, J. R., Dotzlaw, H., and Murphy, L. C., 1996. Estrogen regulation of
nuclear matrix-intermediate filament proteins in human breast cancer cells. J.Cell
Biochem. 63, 174 184
Cowley, S. M., Hoare, S., Mosselman, S., and Parker, M. G., 1997. Estrogen receptors
alpha and beta form heterodimers on DNA. J.Biol.Chem. 272, 19858 19862
Donaldson, S. H., Picher, M., and Boucher, R. C., 2002. Secreted and cell-associated
adenylate kinase and nucleoside diphosphokinase contribute to extracellular
nucleotide metabolism on human airway surfaces. Am.J.Respir.Cell Mol.Biol. 26,
209 215
Dorjsuren, D., Lin, Y., Wei, W., Yamashita, T., Nomura, T., Hayashi, N., and Murakami,
S., 1998. RMP, a novel RNA polymerase II subunit 5-interacting protein,
counteracts transactivation by hepatitis B virus X protein. Mol.Cell Biol. 18, 7546
7555
Dutertre, M. and Smith, C. L., 2000. Molecular mechanisms of selective estrogen
receptor modulator (SERM) action. J.Pharmacol.Exp.Ther. 295, 431 437
Endoh, H., Maruyama, K., Masuhiro, Y., Kobayashi, Y., Goto, M., Tai, H., Yanagisawa,
J., Metzger, D., Hashimoto, S., and Kato, S., 1999. Purification and identification
of p68 RNA helicase acting as a transcriptional coactivator specific for the
activation function 1 of human estrogen receptor alpha. Mol.Cell Biol. 19, 5363
5372
58
Evans, R. M., 1988. The steroid and thyroid hormone receptor superfamily. Science 240,
889 895
Feng, W., Webb, P., Nguyen, P., Liu, X., Li, J., Karin, M., and Kushner, P. J., 2001.
Potentiation of estrogen receptor activation function 1 (AF-1) by Src/JNK through
a serine 118-independent pathway. Mol.Endocrinol. 15, 32 45
Fisher, B., Costantino, J. P., Redmond, C. K., Fisher, E. R., Wickerham, D. L., and
Cronin, W. M., 1994. Endometrial cancer in tamoxifen-treated breast cancer
patients: findings from the National Surgical Adjuvant Breast and Bowel Project
(NSABP) B-14. J Natl.Cancer Inst. 86, 527 537
Fornander, T., Rutqvist, L. E., Cedermark, B., Glas, U., Mattsson, A., Silfversward, C.,
Skoog, L., Somell, A., Theve, T., Wilking, N., and ., 1989. Adjuvant tamoxifen in
early breast cancer: occurrence of new primary cancers. Lancet 1, 117 120
Grabowski, P. J., 1998. Splicing regulation in neurons: tinkering with cell-specific
control. Cell 20;92, 709 712
Harper, M. J. and Walpole, A. L., 1967. A new derivative of triphenylethylene: effect on
implantation and mode of action in rats. J.Reprod.Fertil. 13, 101 119
Jordan, V. C., 2000. Tamoxifen: a personal retrospective. Lancet Oncol. 1, 43 49
Jordan, V. C. and Koerner, S., 1975. Tamoxifen (ICI 46,474) and the human carcinoma
8S oestrogen receptor. Eur.J.Cancer 11, 205 206
59
Kato, S., Endoh, H., Masuhiro, Y., Kitamoto, T., Uchiyama, S., Sasaki, H., Masushige,
S., Gotoh, Y., Nishida, E., Kawashima, H., and ., 1995. Activation of the estrogen
receptor through phosphorylation by mitogen-activated protein kinase. Science
270, 1491 1494
Kirfel, J., Magin, T. M., and Reichelt, J., 2003. Keratins: a structural scaffold with
emerging functions. Cell Mol.Life Sci. 60, 56 71
Klinge, C. M., 2000. Estrogen receptor interaction with co-activators and co-repressors.
Steroids 65, 227 251
Klinge, C. M., 2001. Estrogen receptor interaction with estrogen response elements.
Nucleic Acids Res. 29, 2905 2919
Kuiper, G. G., Enmark, E., Pelto-Huikko, M., Nilsson, S., and Gustafsson, J. A., 1996.
Cloning
of
a
novel
receptor
expressed
in
rat
prostate
and
ovary.
Proc.Natl.Acad.Sci.U.S.A 93, 5925 5930
Kumar, V., Green, S., Stack, G., Berry, M., Jin, J. R., and Chambon, P., 1987. Functional
domains of the human estrogen receptor. Cell 51, 941 951
Labrie, F., Labrie, C., Belanger, A., Simard, J., Gauthier, S., Luu-The, V., Merand, Y.,
Giguere, V., Candas, B., Luo, S., Martel, C., Singh, S. M., Fournier, M., Coquet,
A., Richard, V., Charbonneau, R., Charpenet, G., Tremblay, A., Tremblay, G.,
Cusan, L., and Veilleux, R., 1999. EM-652 (SCH 57068), a third generation
60
SERM acting as pure antiestrogen in the mammary gland and endometrium. J
Steroid Biochem.Mol.Biol. 69, 51 84
Labrie, F., Labrie, C., Belanger, A., Simard, J., Giguere, V., Tremblay, A., and Tremblay,
G., 2001. EM-652 (SCH57068), a pure SERM having complete antiestrogenic
activity in the mammary gland and endometrium. J Steroid Biochem.Mol.Biol.
79, 213 225
Lanz, R. B., McKenna, N. J., Onate, S. A., Albrecht, U., Wong, J., Tsai, S. Y., Tsai, M.
J., and O'Malley, B. W., 1999. A steroid receptor coactivator, SRA, functions as
an RNA and is present in an SRC-1 complex. Cell 97, 17 27
Lee, H., Jiang, F., Wang, Q., Nicosia, S. V., Yang, J., Su, B., and Bai, W., 2000. MEKK1
activation of human estrogen receptor alpha and stimulation of the agonistic
activity of 4-hydroxytamoxifen in endometrial and ovarian cancer cells.
Mol.Endocrinol. 14, 1882 1896
Leone, A., Flatow, U., King, C. R., Sandeen, M. A., Margulies, I. M., Liotta, L. A., and
Steeg, P. S., 1991. Reduced tumor incidence, metastatic potential, and cytokine
responsiveness of nm23-transfected melanoma cells. Cell 65, 25 35
Leone, A., Flatow, U., VanHoutte, K., and Steeg, P. S., 1993. Transfection of human
nm23-H1 into the human MDA-MB-435 breast carcinoma cell line: effects on
tumor metastatic potential, colonization and enzymatic activity. Oncogene 8, 2325
2333
61
Lin, K. H., Wang, W. J., Wu, Y. H., and Cheng, S. Y., 2002. Activation of antimetastatic
Nm23-H1 gene expression by estrogen and its alpha-receptor. Endocrinology 143,
467 475
Lonard, D. M., Nawaz, Z., Smith, C. L., and O'Malley, B. W., 2000. The 26S proteasome
is required for estrogen receptor-alpha and coactivator turnover and for efficient
estrogen receptor-alpha transactivation. Mol.Cell 5, 939 948
Luo, S., Sourla, A., Labrie, C., Gauthier, S., Merand, Y., Belanger, A., and Labrie, F.,
1998. Effect of twenty-four-week treatment with the antiestrogen EM-800 on
estrogen-sensitive parameters in intact and ovariectomized mice. Endocrinology
139, 2645 2656
Maciver, S. K. and Weeds, A. G., 1994. Actophorin preferentially binds monomeric
ADP-actin over ATP-bound actin: consequences for cell locomotion. FEBS Lett.
347, 251 256
Mandlekar, S. and Kong, A. N., 2001. Mechanisms of tamoxifen-induced apoptosis.
Apoptosis. 6, 469 477
Martel, C., Labrie, C., Belanger, A., Gauthier, S., Merand, Y., Li, X., Provencher, L.,
Candas, B., and Labrie, F., 1998. Comparison of the effects of the new orally
active antiestrogen EM-800 with ICI 182 780 and toremifene on estrogensensitive parameters in the ovariectomized mouse. Endocrinology 139, 2486 2492
62
Martel, C., Picard, S., Richard, V., Belanger, A., Labrie, C., and Labrie, F., 2000.
Prevention of bone loss by EM-800 and raloxifene in the ovariectomized rat. J
Steroid Biochem.Mol.Biol. 74, 45 56
Matunis, E. L., Matunis, M. J., and Dreyfuss, G., 1993. Association of individual hnRNP
proteins and snRNPs with nascent transcripts. J.Cell Biol. 121, 219 228
Matunis, M. J., Xing, J., and Dreyfuss, G., 1994. The hnRNP F protein: unique primary
structure, nucleic acid-binding properties, and subcellular localization. Nucleic
Acids Res. 22, 1059 1067
Mayeda, A. and Krainer, A. R., 1992. Regulation of alternative pre-mRNA splicing by
hnRNP A1 and splicing factor SF2. Cell 68, 365 375
McKenna, N. J., Lanz, R. B., and O'Malley, B. W., 1999. Nuclear receptor coregulators:
cellular and molecular biology. Endocr.Rev. 20, 321 344
McKenna, N. J. and O'Malley, B. W., 2000. From ligand to response: generating
diversity in nuclear receptor coregulator function. J Steroid Biochem.Mol.Biol.
74, 351 356
Mosselman, S., Polman, J., and Dijkema, R., 1996. ER beta: identification and
characterization of a novel human estrogen receptor. FEBS Lett. 19;392, 49 53
Mueller, S. O. and Korach, K. S., 2001. Estrogen receptors and endocrine diseases:
lessons from estrogen receptor knockout mice. Curr.Opin.Pharmacol. 1, 613 619
63
Nagaoka, R., Abe, H., Kusano, K., and Obinata, T., 1995. Concentration of cofilin, a
small actin-binding protein, at the cleavage furrow during cytokinesis. Cell
Motil.Cytoskeleton 30, 1 7
Nakamori, S., Ishikawa, O., Ohigashi, H., Imaoka, S., Sasaki, Y., Kameyama, M.,
Kabuto,
T.,
Furukawa,
Clinicopathological
H.,
features
Iwanakga,
and
T.,
prognostic
and
Kimura,
significance
of
N.,
1993.
nucleoside
diphosphate kinase/nm23 gene product in human pancreatic exocrine neoplasms.
Int.J.Pancreatol. 14, 125 133
Nakielny, S. and Dreyfuss, G., 1997. Nuclear export of proteins and RNAs.
Curr.Opin.Cell Biol. 9, 420 429
Nawaz, Z., Lonard, D. M., Dennis, A. P., Smith, C. L., and O'Malley, B. W., 1999.
Proteasome-dependent
degradation
of
the
human
estrogen
receptor.
Proc.Natl.Acad.Sci.U.S.A 96, 1858 1862
Nuber, U., Schwarz, S., Kaiser, P., Schneider, R., and Scheffner, M., 1996. Cloning of
human ubiquitin-conjugating enzymes UbcH6 and UbcH7 (E2-F1) and
characterization of their interaction with E6-AP and RSP5. J.Biol.Chem. 271,
2795 2800
Ogawa, S., Inoue, S., Watanabe, T., Hiroi, H., Orimo, A., Hosoi, T., Ouchi, Y., and
Muramatsu, M., 1998. The complete primary structure of human estrogen
64
receptor beta (hER beta) and its heterodimerization with ER alpha in vivo and in
vitro. Biochem.Biophys.Res.Commun. 243, 122 126
Ostareck, D. H., Ostareck-Lederer, A., Wilm, M., Thiele, B. J., Mann, M., and Hentze,
M. W., 1997. mRNA silencing in erythroid differentiation: hnRNP K and hnRNP
E1 regulate 15-lipoxygenase translation from the 3' end. Cell 89, 597 606
Paramio, J. M. and Jorcano, J. L., 2002. Beyond structure: do intermediate filaments
modulate cell signalling. Bioessays 24, 836 844
Pettersson, K., Grandien, K., Kuiper, G. G., and Gustafsson, J. A., 1997. Mouse estrogen
receptor beta forms estrogen response element-binding heterodimers with
estrogen receptor alpha. Mol.Endocrinol. 11, 1486 1496
Powers, C. A., Mathur, M., Raaka, B. M., Ron, D., and Samuels, H. H., 1998. TLS
(translocated-in-liposarcoma) is a high-affinity interactor for steroid, thyroid
hormone, and retinoid receptors. Mol.Endocrinol. 12, 4 18
Pratt, W. B. and Toft, D. O., 1997. Steroid receptor interactions with heat shock protein
and immunophilin chaperones. Endocr.Rev. 18, 306 360
Ramachandran, C., Catelli, M. G., Schneider, W., and Shyamala, G., 1988. Estrogenic
regulation of uterine 90-kilodalton heat shock protein. Endocrinology 123, 956
961
65
Shyamala, G., Gauthier, Y., Moore, S. K., Catelli, M. G., and Ullrich, S. J., 1989.
Estrogenic regulation of murine uterine 90-kilodalton heat shock protein gene
expression. Mol.Cell Biol. 9, 3567 3570
Simard, J., Labrie, C., Belanger, A., Gauthier, S., Singh, S. M., Merand, Y., and Labrie,
F., 1997. Characterization of the effects of the novel non-steroidal antiestrogen
EM-800 on basal and estrogen-induced proliferation of T-47D, ZR-75-1 and
MCF-7 human breast cancer cells in vitro. Int.J Cancer 73, 104 112
Smith, C. L., DeVera, D. G., Lamb, D. J., Nawaz, Z., Jiang, Y. H., Beaudet, A. L., and
O'Malley, B. W., 2002. Genetic ablation of the steroid receptor coactivatorubiquitin ligase, E6-AP, results in tissue-selective steroid hormone resistance and
defects in reproduction. Mol.Cell Biol. 22, 525 535
Smith, C. L., Nawaz, Z., and O'Malley, B. W., 1997. Coactivator and corepressor
regulation of the agonist/antagonist activity of the mixed antiestrogen, 4hydroxytamoxifen. Mol.Endocrinol. 11, 657 666
Sourla, A., Luo, S., Labrie, C., Belanger, A., and Labrie, F., 1997. Morphological
changes induced by 6-month treatment of intact and ovariectomized mice with
tamoxifen and the pure antiestrogen EM-800. Endocrinology 138, 5605 5617
Steeg, P. S., Bevilacqua, G., Kopper, L., Thorgeirsson, U. P., Talmadge, J. E., Liotta, L.
A., and Sobel, M. E., 1988. Evidence for a novel gene associated with low tumor
metastatic potential. J.Natl.Cancer Inst. 80, 200 204
66
Tang, P. Z., Gannon, M. J., Andrew, A., and Miller, D., 1995. Evidence for oestrogenic
regulation of heat shock protein expression in human endometrium and steroidresponsive cell lines. Eur.J.Endocrinol. 133, 598 605
Terenius, L., 1971. Structure-activity relationships of anti-oestrogens with regard to
interaction with 17-beta-oestradiol in the mouse uterus and vagina. Acta
Endocrinol.(Copenh) 66, 431 447
Tora, L., White, J., Brou, C., Tasset, D., Webster, N., Scheer, E., and Chambon, P., 1989.
The human estrogen receptor has two independent nonacidic transcriptional
activation functions. Cell 59, 477 487
Tremblay, G. B., Tremblay, A., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., Labrie, F.,
and Giguere, V., 1997. Cloning, chromosomal localization, and functional
analysis of the murine estrogen receptor beta. Mol.Endocrinol. 11, 353 365
Tsai, M. J. and O'Malley, B. W., 1994. Molecular mechanisms of action of
steroid/thyroid receptor superfamily members. Annu.Rev.Biochem. 63:451-86.,
451 486
Walinder, O., 1968. Identification of a phosphate-incorporating protein from bovine liver
as nucleoside diphosphate kinase and isolation of 1-32P-phosphohistidine, 3-32Pphosphohistidine, and N-epsilon-32P-phospholysine from erythrocytic nucleoside
diphosphate kinase, incubated with adenosine triphosphate-32P. J.Biol.Chem.
243, 3947 3952
67
Walter, S. and Buchner, J., 2002. Molecular chaperones--cellular machines for protein
folding. Angew.Chem.Int.Ed Engl. 41, 1098 1113
Webb, P., Nguyen, P., Shinsako, J., Anderson, C., Feng, W., Nguyen, M. P., Chen, D.,
Huang, S. M., Subramanian, S., McKinerney, E., Katzenellenbogen, B. S.,
Stallcup, M. R., and Kushner, P. J., 1998. Estrogen receptor activation function 1
works by binding p160 coactivator proteins. Mol.Endocrinol. 12, 1605 1618
Weissman, A. M., 2001. Themes and variations on ubiquitylation. Nat.Rev.Mol.Cell
Biol. 2, 169 178
Willems, R., Slegers, H., Rodrigus, I., Moulijn, A. C., Lenjou, M., Nijs, G., Berneman, Z.
N., and Van Bockstaele, D. R., 2002. Extracellular nucleoside diphosphate kinase
NM23/NDPK modulates normal hematopoietic differentiation. Exp.Hematol. 30,
640 648
Wu, W. X., Derks, J. B., Zhang, Q., and Nathanielsz, P. W., 1996. Changes in heat shock
protein-90 and -70 messenger ribonucleic acid in uterine tissues of the ewe in
relation
to
parturition
and
regulation
by
estradiol
and
progesterone.
Endocrinology 137, 5685 5693
Yan, F., Gao, X., Lonard, D. M., and Nawaz, Z., 2003. Specific ubiquitin-conjugating
enzymes promote degradation of specific nuclear receptor coactivators.
Mol.Endocrinol. 17, 1315 1331
68
Figure legends
Figure 1. The effect of estradiol and SERMs on gene expression and proliferation in
Ishikawa cells. (A) Ishikawa cells (2 x 105 cells/well) were transfected with 500 ng of
ERE2e1b-luciferase reporter and 50 ng of expression vector for hERα. 24 hours after
transfection, cells were incubated with vehicle (lane 1, Veh) 10-8 M estradiol (lane 2, E2),
10-7 M 4-hydroxytamoxifen (lane 3, 4-OHT) or 10-7 M EM652 (lane 4) for 24 hours.
Standard luciferase assays were performed on cell extracts as described in the Materials
and Methods and the mean value of triplicate samples ± S.D. are plotted. (*) = P<.05. (B)
The effect of estradiol and SERMs on endogenous ER-dependent genes. Ishikawa cells (2
x 106 cells/plate) were incubated with vehicle (lane 1) 10-8 M estradiol (lane 2), 10-7 M 4hydroxytamoxifen (lane 3) or 10-7 M EM-652 (lane 4) for 4 hours. Expression of the
lactoferrin and progesterone receptor genes was measured by real time RT-PCR as
described under Materials and Methods. Expression was normalized to GAPDH, and
values were plotted relative to vehicle treated samples (set to 1). Each bar represents the
mean value ± S.D. (*): P<.05. (C) Growth response of Ishikawa cells following
incubation with estradiol and SERMs. Ishikawa cells incubated with vehicle (lane 1), 10-8
M estradiol (lane 2), 10-7 M 4-hydroxytamoxifen (lane 3), 10-10 M 4-hydroxytamoxifen
(lane 4), 10-7 M EM-652 (lane 5), and 10-10 M EM-652 (lane 6) for 9 days as described in
the Materials and Methods. Cell growth was assessed by the MTT assay. Each bar
represents the mean value ± S.D. (*) = P<.05.
Figure 2. Two-dimensional gel images of total cellular proteins from Ishikawa cells
incubated with estradiol and SERMs. Ishikawa cells (5 x 106 cell/plate) were incubated
69
-8
-7
for 24 hours with (A) vehicle, (B) 10 M estradiol, (C) 10 M 4-hydroxytamoxifen or
-7
(D) 10 M EM-652. 500 µg of total cellular protein was separated in the first dimension
by isoelectric focussing using IPG pH 3-10 NL 2D PAGE (12.5% T), and the 2nd
dimension by 12% SDS-PAGE. The proteins were visualized by colloidal Coomassie
blue stain. The regions enlarged in Fig. 3 are boxed and numbered 1-4.
Figure 3. Enlarged regions of the two-dimensional gel images of total cellular lysate from
Figure 2. The dotted boxes indicate no change in protein expression relative to vehicle;
solid boxes indicate a change in protein expression relative to vehicle (A) Enlarged gel
image corresponding to region 1 in Figure 2. Induction of HSP 90 α & β following 24
hour incubation with 10
-8
-7
M estradiol and 10 M EM-652. (B) Enlarged gel image
corresponding to region 2 in Figure 2. Induction of CK18 and hnRNP F after 24 hour
-8
-7
incubation with 10 M estradiol and 10 M 4-hydroxytamoxifen. (C) Enlarged gel image
corresponding to region 3 in Figure 2. Induction of CK8 after 24 hour incubation with 10
8
-
-7
M estradiol and 10 M 4-hydroxytamoxifen. (D) Enlarged gel image corresponding to
region 1 in Figure 2. Induction of cofilin, UBCh7, and RBP5-mediating protein after 24
-7
incubation with 10 M EM-652.
Figure 4. Two-dimensional gel images of secreted proteins from Ishikawa cells.
Ishikawa cells (5 x 106 cell/plate) were cultured in serum-free DMEM and incubated for
-8
-7
24 hours with (A) vehicle, (B) 10 M estradiol, (C) 10 M 4-hydroxytamoxifen, or (D)
-7
10 M EM-652. The secreted proteins were isolated as described under Materials and
Methods. 100 µg of protein was separated in the first dimension by isoelectric focussing
using IPG pH 3-10 NL 2D PAGE (12.5% T) and in the 2nd dimension by 12% SDS70
PAGE. Proteins were visualized by silver stain. The region enlarged in panel E is boxed
and numbered 1. (E) Enlarged gel image corresponding to region 1 in Figure 2 Panel A,
-8
-7
B C, D. Induction of NDPK B after 24 hour incubation with 10 M estradiol and 10 M
4-hydroxytamoxifen..
Figure 5. Western blot analysis of estradiol and SERM regulated proteins. (A) Following
-8
-7
-7
a 24 incubation with vehicle, 10 M estradiol, 10 M 4-hydroxytamoxifen or 10 M EM652, total cellular protein extract was prepared from Ishikawa cells for Western blotting
analysis membrane as described in the Material and Methods. The membranes were
probed with antibodies against hnRNP F, NDPK B, Ck8/18, cofilin, HSP90 and UBCh7.
Representative Western blots from one of three separate experiments. (B).Quantitation of
the Western blot signal normalized to β-actin. The graphs indicate the fold difference of
each protein expression versus vehicle (set at a value of 1) following measurement of
Western blot signal intensity by Kodak Image Station 440CF and 1D Image Analysis
software. In each independent experiment, duplicate samples for each treatment condition
were normalized to the β-actin levels. The experiment was performed three times and the
mean values from these experiments ± S.D. is reported in the graphs. Asterisk indicates
statistically significant difference from vehicle treatment analyzed by one-way ANOVA
Dunnett’s T Test. (*p<0.05).
71
Table 1.
Identification of proteins regulated by estradiol and SERMs in whole cell lysate
Proteins
Keratin type I
cytoskeletal 18 (ck18)
Heterogeneous nuclear
ribonucleoprotein F
(hnRNP F)
Keratin type ii
cytoskeletal 8 (ck 8)
Ubiquitin-conjugating
enzyme E2-18kDa
(UbcH7)
RNA polymerase II
subunit 5-mediating
protein
Cofilin, non-muscle
isoform
Hsp 90-α
Hsp 90-β
MW
(kDa)
pI
Con
Est
Tam
EM652
#Peptides
Sequenced
Accession #
47.9
5.34
⎯
↑
↑
⎯
4
P05783
45.6
5.38
⎯
↑
↑
⎯
4
P52597
53.5
5.52
⎯
↑
↑
⎯
5
P057897
17.8
8.7
⎯
⎯
⎯
↑
3
P51966
57.6
4.96
⎯
⎯
⎯
↑
2
O94763
18.5
8.22
⎯
⎯
⎯
↑
4
P23528
84.5
4.9
⎯
↑
⎯
↑
2
P07900
83.1
4.9
⎯
↑
⎯
↑
3
P08238
Identification of proteins regulated by estradiol and SERMs in the secreted fraction
Nucleoside Diphosphate
Kinase B
17.3
8.52
⎯
↑
Est = estradiol
Tam = 4-hydroxytamoxifen
Accession# according to SwissProt database
↑ = Increase in expression relative to control
– = No change relative to control
72
↑
⎯
2
P22392
A
C
3.5
*
2.5
0.30
*
2.0
*
0.20
1.5
*
1.0
*
0.25
O.D.
Fold Induction
3.0
0.35
0.15
0.10
0.05
0.5
0.0
Veh
E2
0.00
4-OHT EM652
4-OHT
E2
Veh
8
*
10
8
6
*
4
Fold Induction
12
Fold Induction
10-10M
10-7M
10-10M
10-7M
Progesterone
Receptor
Lactoferrin
B
EM652
*
6
*
4
2
2
0
0
Veh
E2
4-OHT EM652
Veh
E2 4-OHT EM652
Fig. 1
73
A. Vehicle
SDS-PAGE
3
B.
Estradiol
10
pH
1
1
3
2
3
2
4
4
1
1
2
3
2
3
4
4
C. Tamoxifen
D. EM-652
Fig. 2
74
A.
V eh
E2
4 -O H T
B.
C K 18
hnR N PF
C.
CK 8
E M 652
H SP 90 α & β
D.
C o filin
U b cH 7
R B P 5 m ed ia tin g
P ro tein
F ig. 3
75
A. Vehicle
Estradiol
10
pH
SDS-PAGE
3
B.
1
1
1
1
C. Tamoxifen
E.
Veh
D.
EM652
E2
4-OHT
EM652
NDPK B
Fig. 4
76
Veh
A.
4-OHT
E2
EM 652
hnRNP F
NDPKB
CK 8/18
Cofilin
HSP90
UBCh7
ß-Actin
NDPK B
0.5
*
6
4
2
EM652
UBCh7/ ß-actin
1.0
4-OHT
8
1.5
E2
UBCh7
10
2.5
2.0
Veh
1
2
* *
3.0
EM652
2
4
4-OHT
*
*
6
E2
*
3
EM652
4-OHT
HSP90
4
CK8/18/ ß-actin
4
2
Ck8/18
*
8
Veh
NDPK B/ ß-actin
*
6
E2
HSP90/ ß-actin
*
12
10
8
Veh
hnRNP F/ ß-actin
hnRNP F
Cofilin
3.0
*
2.5
2.0
1.5
1.0
0.5
EM652
4-OHT
E2
Veh
EM652
4-OHT
E2
Veh
EM652
4-OHT
E2
Veh
Cofilin/ ß-actin
B.
Fig. 5
77
Veh
E2
4-OHT
EM 652
C.
*
*
4-OHT
5
4
E2
NDPK B
6
3
2
1
EM652
Veh
NDPK B/ Cell No#
NDPKB (secreted)
Fig. 5
78
Title: The Src Kinase Pathway Promotes Tamoxifen Agonist Action in Ishikawa
Endometrial Cells through Phosphorylation-Dependent Stabilization of Estrogen
Receptor α Promoter Interaction and Elevated SRC-1 Activity.
Abbreviated Title: Src Kinase Potentiation of Tamoxifen Agonist Action
Yatrik M. Shah and Brian G. Rowan∗
Department of Biochemistry & Molecular Biology
Medical College of Ohio, Toledo, OH
∗
Corresponding author:
Brian G. Rowan, Ph.D.
Department of Biochemistry & Molecular Biology
Medical College of Ohio
3035 Arlington Ave.
Toledo, OH 43614-5804
Tel#: 419-383-4134
Fax#: 419-383-6228
Email: [email protected]
Keywords: Estrogen Receptor, Endometrium, Tamoxifen, Src Kinase
79
Abstract
Tamoxifen is the most widely used selective estrogen receptor modulator (SERM) for
breast cancer in clinical use today. However, tamoxifen agonist action in endometrium
remains a major hurdle for tamoxifen therapy. Activation of the non-receptor tyrosine
kinase src promotes tamoxifen agonist action, although the mechanisms remain unclear.
To examine these mechanisms, the effect of src kinase on estrogen and tamoxifen
signaling in tamoxifen-resistant Ishikawa endometrial adenocarcinoma cells was
assessed. A novel connection was identified between src kinase and serine 167
phosphorylation in estrogen receptor-α (ER) via activation of AKT kinase. Serine 167
phosphorylation stabilized ER interaction with endogenous ER-dependent promoters.
Src kinase exhibited the additional function of potentiating the transcriptional activity of
Gal-SRC-1 and Gal-CREB binding protein (CBP) in endometrial cancer cells while
having no effect on Gal-PCAF and Gal fusions of the other p160 coactivators GRIP1
(TIF-2/NcoA-2/SRC-2) and AIB-1 (RAC-3/ACTR/SRC-3). Src effects on ER
phosphorylation and SRC-1 activity both contributed to tamoxifen agonist action on ERdependent gene expression in Ishikawa cells. Taken together, these data demonstrate that
src kinase potentiates tamoxifen agonist action through serine 167-dependent
stabilization of ER promoter interaction and through elevation of SRC-1 and CBP
coactivation of ER.
80
Introduction
Estrogen Receptor-α (referred to as ER) is a member of the nuclear receptor
superfamily of transcription factors that is activated by ligand binding of estrogenic and
other ligands. ER is also activated by ligand-independent mechanisms that involve
crosstalk from peptide and growth factor signal transduction pathways. In the liganddependent pathway, estrogen crosses the cell membrane and binds to the ligand-binding
domain (LBD) of the ER. Upon ligand binding, receptor interaction with heat shock
protein-90 (HSP-90) and other chaperone proteins is lost and the ER dimerizes and binds
to estrogen responsive elements (ERE) in the promoters of ER-dependent genes (1;2). ER
activation results in recruitment of coactivators of the p160 family (SRC-1 (NcoA-1)
(3;4), GRIP1 (TIF-2/NcoA-2/SRC-2) (5;6), AIB-1 (RAC-3/ACTR/SRC-3) (7-11)) and
CREB binding protein (CBP) (12) that modify chromatin structure and facilitate mRNA
transcription (13).
Both ligand-dependent and ligand-independent activation of ER is modulated by
receptor phosphorylation. ER is a phosphoprotein and upon ligand binding receptor
phosphorylation is enhanced (14-16). The major phosphorylation sites of ER reside in the
N-terminal domain (NTD) at serines 104, 106, 118, and 167. Mutation of serines 104,
106, and 118 to alanine results in a general decrease in ER transcriptional activity (14).
Phosphorylation at serine 167 was shown to be important in DNA binding by the receptor
(17-19). Numerous signaling pathways regulate ER phosphorylation. Serines
104/106/118 are targets of the mitogen activated protein kinases (MAPK) family (20-22),
and serine 167 has been shown to be phosphorylated by AKT (22-25).
81
In addition to ER, cellular signaling pathways phosphorylate coregulator proteins.
SRC-1 is phosphorylated on seven sites in vivo all of which contain a consensus sequence
for proline-directed protein kinases (26). In addition, protein kinase A (PKA) enhanced
ligand-independent activation of PR through phosphorylation of SRC-1 (27). GRIP-1
and AIB-1 are phosphorylated in vitro by MAP kinases (28;29). In breast cancer cells the
transcriptional activity of AIB-1 is increased by MAPK phosphorylation which stimulates
the recruitment of histone acetyltransferases (28). In addition to coactivators, cellular
signaling cascades also phosphorylate corepressors. Interaction of N-CoR and SMRT
with PR decreased after incubation with 8-bromo cAMP (30). In vitro phosphorylation
by MEKK decreased the interaction of SMRT with thyroid receptor (31). Interaction of
the corepressor N-CoR with the ER is decreased by treatment with forskolin and
epidermal growth factor (32).
Coregulator interactions with ER are important mechanisms mediating selective
estrogen receptor modulator (SERM) action. Tamoxifen inhibits coactivator recruitment
and promotes corepressor association with ER to inhibit estrogen-dependent gene
transcription in tamoxifen-sensitive breast cancer cells (33). With regard to this
mechanism, elevation of coactivator proteins and reduction in corepressor proteins may
be associated with the progression of breast cancer to a more malignant and tamoxifenresistant phenotype. In a mouse model for human breast cancer, decreased levels of NCoR correlated with the onset of resistance to tamoxifen therapy (32). Jepsen et. al. (34)
showed that the absence of N-CoR in mouse embryo fibroblasts isolated from the N-CoR
null mouse allowed 4-hydroxytamoxifen to act as a full agonist for ER-dependent
82
transcription. 4-hydroxytamoxifen did not activate ER-dependent transcription in wildtype mouse embryo fibroblasts containing normal N-CoR levels. The hypothesis that
elevated coactivator levels promote tamoxifen action may be relevant in the uterus since
tamoxifen is an agonist in normal uterus (35;36) and endometrial-derived cell lines (3739). However in contrast to normal versus malignant breast, we did not detect any distinct
differences between coactivator and corepressor expression patterns in endometrium with
both coactivator and corepressor levels increased during the progression from normal to
malignant endometrium (40).
Several reports suggest activated cellular signaling pathways in the uterus may
promote the tissue specific agonist effects of tamoxifen. Phosphorylation at serine 118 in
ER increased transcriptional activation in response to estrogen and tamoxifen (21).
MEKK1 increased the agonistic activity of tamoxifen implicating downstream MAPKs in
regulating tamoxifen agonist/antagonist action (41). Src kinase can enhance the
transcriptional activity of the tamoxifen-ER complex through the MAPK pathway (42).
Finally, although elevated Src kinase activity is detected in late stage steroid resistant
breast cancer (43) we recently found that src kinase activity is higher in normal compared
to malignant endometrium (44). This may indicate a role for activated Src kinase in
promoting tamoxifen agonist action in normal endometrium
Src kinase is a non-receptor tyrosine kinase and a proto-oncogene that is
deregulated in several different cancers (45). Src kinase activates several signaling
cascades including the MAPK and AKT pathways (46-48) both of which impact on ERdependent signaling. Feng et al. (42) reported two independent pathways for src kinase
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potentiation of estrogen and tamoxifen signaling in HeLa cells: 1) an ER-dependent
pathway that is partially dependent on phosphorylation of serine 118 and; 2) an ERindependent pathway implicating src kinase effects on coregulators. However, the
precise mechanisms and specific phosphorylation sites through which src kinase
modulates ER signaling are incompletely defined, most likely because multiple proteins
important for ER signaling are targeted by src. For a more thorough, mechanistic
understanding of the role of src kinase in tamoxifen agonist action in the endometrium,
the effect of src on both ER and coactivator activity and phosphorylation was
investigated. This report describes src kinase induced phosphorylation of ER serine 167
via AKT kinase as a major mechanism for src potentiation of ER signaling through
stabilization of ER promoter interaction. Src kinase also specifically elevated SRC-1
activity through multiple phosphorylation sites. Finally, cell-type specific targeting of
CBP by src kinase was demonstrated.
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Results
Src kinase modulates estradiol and 4-hydroxytamoxifen action on ERE2e1bluciferase reporter in an endometrial-derived cell line. Interest in src kinase
potentiation of tamoxifen agonist activity in endometrium stemmed from our observation
that tamoxifen-resistant endometrial cancer cell lines (Ishikawa and HEC1A) exhibited
elevated active src compared to tamoxifen-sensitive breast cancer cell lines (MCF-7 and
T47D) (Fig. 1A). To further confirm the role of endometrial src kinase in tamoxifen
agonism, Ishikawa cells were transfected with an ERE2e1b-luciferase reporter and either
incubated with SU6656, a specific src kinase inhibitor (Fig. 1B) or cotransfected with a
dominant active mutant of src kinase (Fig. 1C) followed by measurement of luciferase
expression. Incubation of Ishikawa cells with 4-hydroxytamoxifen resulted in similar
activation of the reporter (1.5 –2 fold relative to vehicle) as occurred following
incubation of cells with estradiol (3-4 fold relative to vehicle) (Fig. 1B). Both 4hydroxytamoxifen and estradiol activation of the reporter was significantly reduced in
cells incubated with SU6656 (Fig. 1B, lanes 4 and 6) These data coincided with reduced
src kinase activation in cells incubated with SU6656 as measured by Western blot
analysis using a phospho-specific src kinase antibody (data not shown). Transfection
with dominant active src kinase increased basal luciferase expression almost two fold
indicating ligand-independent activation of ER (Fig.1C, lane 2). Dominant active src also
potentiated estradiol and 4-hydroxytamoxifen stimulated luciferase expression 4-5 fold
and 3 fold relative to vehicle, respectively (Fig 1C, lanes 4 and 6)). To confirm that the
inhibition of src kinase downregulates estradiol and 4-hydroxytamoxifen activation of the
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ERE2e1b-luciferase reporter, siRNA was used to reduce src protein levels. Western blot
analysis confirmed that src protein levels were reduced more than 65% in Ishikawa cells
transfected with siRNA for src kinase (siRNA-src) (Fig. 1D). When siRNA-src was
cotransfected with ERE2e1b-luciferase reporter, the basal activity was reduced by more
than 50% (Fig. 1E, lane 2) and both the estradiol and 4-hydroxytamoxifen induction of
the reporter was decreased to levels equivalent to vehicle alone (Fig. 1E, lanes 4 and 6 vs.
lane 1). To rule out nonspecific effects of SU6656, siRNA-src and dominant active c-src
on general transcription, these agents were incubated with or cotransfected into cells with
a constitutively active RSV-luciferase reporter (Fig. 1F). These agents had no significant
effect on the RSV-luciferase reporter suggesting that src kinase specifically modulates
estradiol and 4-hydroxytamoxifen signaling. The requirement of ER for the src kinase
effects on estradiol and 4-hydroxytamoxifen signaling was demonstrated by incubation of
Ishikawa cells with the pure antiestrogen ICI 182,780. ICI 182,780 blocked dominant
active src potentiation of estradiol and 4-hydroxytamoxifen activation of the reporter
(data not shown). Furthermore as previously reported (42), dominant active src
potentiated estradiol and 4-hydroxytamoxifen activation of ERE2e1b-luciferase only in
cells cotransfected with ER (data not shown).
Src kinase modulates estradiol and 4-hydroxytamoxifen action on endogenous ERregulated genes. Real time RT-PCR was used to measure expression of two wellcharacterized ER-regulated genes, c-myc and pS2. The c-myc gene is regulated by ER
through both a consensus ERE as well as a nonconsensus sequence hypothesized to be an
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AP1 site. ER indirectly associates with the AP-1 site through direct binding with AP1
proteins (33;49). The pS2 gene contains consensus EREs in the 5’ flanking region of the
gene (50). C-myc and pS2 gene expression were measured following incubation with
estradiol and 4-hydroxytamoxifen alone or in combination with the src kinase inhibitor
SU6656. Both estradiol and 4-hydroxytamoxifen induced c-myc gene expression 2-3
fold and 4-5 fold relative to vehicle, respectively (Fig. 2A, lanes 3 and 5). The pS2 gene
was regulated by ligands in a similar manner (Fig. 2B). Ligand dependent induction of cmyc and pS2 was reduced more than 50% by coincubation of cells with SU6656 (Fig.
2A, B lanes 4 and 6).
Cells that exhibit high transfection efficiency were needed to measure the role of
dominant active src on endogenous ER-regulated genes. Although the Ishikawa cells
were not appropriate for these experiments due to low transfection efficiency, the wellcharacterized ER-negative HeLa cells exhibited more than 90% transfection efficiency
(data not shown) and were therefore useful to measure effects of both dominant active src
and various ERα mutants. It should be noted that unlike Ishikawa cells, HeLa cells
transfected with ER expression plasmid do not exhibit tamoxifen agonist activity. High
efficiency transfection of HeLa cells with ER was sufficient to initiate estradiol, but not
4-hydroxytamoxifen, induction of endogenous c-myc and pS2 gene expression 6-7 fold
and 4-5 fold relative to vehicle, respectively (supplemental data Fig. S-1A, S-1B). Similar
to results in Ishikawa cells, cotransfection of HeLa cells with dominant active src
potentiated basal, and estradiol-regulated c-myc and pS2 expression. Remarkably,
although 4-hydroxytamoxifen alone had no effect on gene expression and dominant
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active src alone resulted in only 1.5 fold induction over vector transfected cells, the
combination of tamoxifen and dominant active src induced c-myc and pS2 gene
expression to a level comparable to estradiol alone (supplemental data Fig. S-1). These
data demonstrate that src kinase promotes tamoxifen agonist action on endogenous genes
in a cellular context in which tamoxifen is an antagonist. Although, HeLa and Ishikawa
cells differ with respect to tamoxifen agonism/antagonism, ERα negative Ishikawa
variants transfected with ER do not exhibit tamoxifen agonist activity and in this regard
are similar to HeLa cells (51). Although tamoxifen agonism/antagonism cannot be
directly compared in HeLa and Ishikawa cells, it is clear that for each line activation of
the src pathway potentiates tamoxifen agonist activity above control.
Src kinase partially mediates its effect on ER-dependent transcription through
phosphorylation at both serine 118 and serine 167. As previously reported (42), src
kinase-mediated effects on ER-regulated gene expression was localized to the NTD of
ER (data not shown). Consequently, phosphorylation within this domain was measured
using phospho-specific antibodies for serine 104/106, 118, and 167. In ER-transfected
HeLa cells, estradiol increased phosphorylation at serines 118 and 167, an effect that was
inhibited by co-incubation with SU6656 (Fig. 3A). A band was not detected when
Western blots were probed with the antibody specific for phospho-104/106 on ER (data
not shown). Similar results were found in Ishikawa cells in which both estradiol and 4hydroxytamoxifen consistently increased serine 118 and serine 167 phosphorylation, an
effect that was blocked by co-incubation with SU6656 (data not shown). To further
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confirm these results, point mutations of each phosphorylation site were prepared and
cotransfected with ERE2e1b-luciferase into ER-negative HeLa cells to assess whether
dominant active src could potentiate estradiol-mediated induction of the reporter. As
previously described (14), mutation of the ER NTD phosphorylation sites decreased basal
and estradiol-stimulated reporter activation (Fig. 3B) although the mutations did not
affect ER protein levels (Fig. 3C). Dominant active src potentiated estradiol-stimulated
luciferase expression by wild type ER, ER/S104A, and ER/S106A mutants, but not the
ER/S118A and ER/S167A mutants (Fig. 3B). S118A and ER/S167A also blocked the
ability of dominant active src to induce tamoxifen activation of ERE2e1b-luciferase in
HeLa cells (data not shown). Taken together, these data demonstrate that src kinase can
mediate effects on ER-dependent gene transcription through phosphorylation of serine
118 as previously described (42) and also through phosphorylation at serine 167.
Inhibition of src kinase disrupts interaction of ER with endogenous promoters.
Previous work has established that src kinase increases serine 118 phosphorylation
through activation of the ERK1/2 pathway in HeLa cells (42). However, there are no
reports of src kinase modulating phosphorylation of ER serine 167 and no studies have
examined regulation of ER signaling in endometrial-derived, tamoxifen resistant cells.
Because serine 167 is reported to regulate in vitro DNA binding by ER (17-19), the role
of src kinase in ER interaction with endogenous promoters was assessed by ChIP assays.
To assess several paradigms of ER-dependent gene expression, three promoter elements
were examined: 5’ consensus ERE on the pS2 promoter, the consensus ERE on the c-myc
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promoter (52), and the nonconsensus ERE on the c-myc promoter (33). In Ishikawa cells,
estradiol and 4-hydroxytamoxifen increased ER recruitment to the ERE sequence in the
pS2 promoter and this ligand-dependent recruitment was blocked by co-incubation with
src kinase inhibitor SU6656 (Fig. 4, left panel). Conversely, SU6656 did not affect the
well-characterized interaction of SP1 with the hTERT promoter (53) demonstrating that
the effect of SU6656 on ER was not a result of general inhibition of transcription factor
binding to DNA (data not shown). Quantitative ChIP assays verified the increased
promoter recruitment of ER by estradiol and 4-hydroxyamoxifen and the inhibition of
this recruitment by SU6656 (Fig. 4, right panel). Identical results to those described in
Figure 4 were found for the consensus ERE and the nonconsensus ERE on the c-myc
promoter (Supplemental data, Fig. S-2). These data indicate that src kinase modulates
ligand-dependent ER promoter recruitment. Interestingly, estradiol resulted in
quantitatively greater promoter recruitment of ER than 4-hydroxytamoxifen, an
observation that has not previously been reported.
Src kinase partially mediates its effect on ER-dependent transcription through
PI3K/AKT kinase. Inhibition of AKT disrupts ER promoter interaction. Since src
mediates its effects on ER through serine 167 phosphorylation, it was important to
identify the downstream effectors of src that lead to altered serine 167 phosphorylation.
Although src has been shown to activate AKT pathways (48) and in vitro
phosphorylation of serine 167 by AKT has been reported (23-25), the connection
between src kinase and serine 167 phosphorylation has not been demonstrated.
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Consequently it was important to assess whether AKT could mediate src effects on ERdependent gene transcription in Ishikawa cells. Western blot analysis revealed an
increase in phospho-AKT when cells were incubated with estradiol and 4hydroxytamoxifen (Fig. 5A left and right panel, lanes 3 and 5) and this induction was
blocked by co-incubation with SU6656 (Fig. 5A, left and right panel, lanes 4 and 6). To
determine whether AKT inhibition would block the dominant active src potentiation of
estradiol- and 4-hydroxytamoxifen-mediated gene transcription, Ishikawa cells were
incubated with either a specific AKT inhibitor or an inhibitor of phosphoinositide 3-OH
kinase (PI3K), a kinase upstream of AKT. Ishikawa cells were cotransfected with
ERE2e1b-luciferase and dominant active src and incubated with wortmannin (PI3K
inhibitor) or an AKT inhibitor alone or in combination with estradiol or 4hydroxytamoxifen. Incubation with wortmannin or the AKT inhibitor completely
blocked dominant active src potentiation of the estradiol and 4-hydroxytamoxifen
stimulated ERE2e1b-luciferase (Fig. 5B and C, lanes 8 and 12) suggesting that src kinase
signals to ER through AKT. Control Western blots indicated that AKT activity was
inhibited following incubation with PI3K inhibitor or the AKT inhibitor and neither agent
affected the constitutively active RSV-luciferase reporter in transfected Ishikawa cells
(data not shown). To determine if inhibition of AKT alters ER interaction with
endogenous promoters, ChIP assays were performed in Ishikawa cells on the three
promoter elements described in Figure 4. The estradiol- and 4-hydroxytamoxifen-induced
recruitment of ER to the ERE sequence in the pS2 was blocked by co-incubation with
wortmannin (Fig. 5D). Conversely, wortmannin did not affect SP1 interaction with the
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hTERT promoter (data not shown). Identical results to those described in Figure 5 were
found for the consensus ERE and the nonconsensus ERE on the c-myc promoter
(Supplemental data, Fig. S-3). Taken together, these data demonstrate that src kinase
potentiates estradiol and 4-hydroxytamoxifen action by modulating ER promoter
interaction via the PI3K/AKT pathway.
Serine 167 of the ER regulates promoter interaction by the receptor. To directly
investigate the role of ER phosphorylation in promoter interaction, quantitative ChIP
assays were performed in ER-negative HeLa cells transfected with ER phosphorylation
site mutants. HeLa cells were cotransfected with dominant active src and either wild type
ER, ER/S118A or ER/S167A and promoter interaction was measured as described above.
There was a decrease in basal and estradiol-stimulated recruitment of ER/S167A to the
pS2 promoter compared to wild type ER and ER/S118A (Fig. 6). A previous report
described a similar result for ER/S167A using in vitro interaction with a synthetic ERE
sequence (19). Dominant active src increased basal level and estradiol-stimulated
recruitment of wild type ER (Fig. 6 lanes 2, 4) and ER/S118A (Fig. 6 lanes 6, 8) to the
pS2 promoter but had no effect on ER/S167A recruitment (Fig. 6 lanes 10, 12). Similar
results were found with the c-myc promoter (Supplemental data Fig. S-4). These results
demonstrate that src kinase modulates ER promoter interaction through phosphorylation
of serine 167. Interestingly, mutation of serine 118 to alanine had no effect on ER
promoter interaction suggesting that mechanisms other than DNA binding are responsible
for regulation of ER signaling by serine 118 phosphorylation.
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Src kinase potentiates SRC-1 and CBP transactivation in endometrial-derived cell
lines. To measure the effects of src kinase on coactivator activity, SRC-1, SRC-2 (TIF2/GRIP-1), SRC-3 (AIB-1/RAC-3/ACTR), PCAF, and CBP were fused to the GAL4DBD expression plasmid and cotransfected with a Gal response element reporter (GalREluciferase) into Ishikawa and HEC-1A cells. All GAL constructs except GAL-PCAF
activated GalRE-luciferase above activity of the GAL-DBD alone. When dominant
active src or siRNA-src was cotransfected with the GAL-coactivator constructs, only
GAL-SRC-1 and GAL-CBP transactivation function was both potentiated by dominant
active src and inhibited by siRNA-src in Ishikawa cells (Fig. 7A). The effect of the src
kinase inhibitor SU6656 on the GAL-coactivator activities was also assessed. The src
inhibitor reduced GAL-SRC-1 and GAL-CBP activity while having no effect on GALSRC-2, GAL-SRC-3, and GAL-PCAF activity (Fig. 7B). These assays were performed
in an additional endometrial-derived cell line, HEC1A. When the GAL-coactivator
constructs were cotransfected with either the dominant active src or siRNA-src, only
SRC-1 activity was potentiated with the dominant active src and repressed with the
siRNA-src (Fig 7C). Src kinase inhibitor SU6656 inhibited the activities of all the GALcoactivator constructs, possibly through nonspecific effects since the GAL-DBD alone
was also inhibited (Fig. 7C). Because src kinase selectively activated the p160 family
coactivator SRC-1, it was of interest to determine whether specific SRC-1
phosphorylation sites were mediating the increased SRC-1 activity. Seven SRC-1
phosphorylation sites (26) were mutated to alanine (S372A, S395A, S517A, S569A,
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S1033A and T1179A/S1185A) and each single phosphorylation site mutant was fused to
the GAL4-DBD. Dominant active src kinase potentiated activity of each single site SRC1 phosphorylation mutant in both Ishikawa and HEC1A cells (data not shown). These
findings argue against the notion that a single SRC-1 phosphorylation site mediates src
kinase potentiation of SRC-1 activity although the possibility of different combinations
of sites was not examined. The preceding data confirm that src kinase specifically
modulates the activity of SRC-1 and CBP in Ishikawa cells. Interestingly, CBP activity
was not altered by src kinase in HEC1A cells suggesting cell specific targeting by src
kinase (Fig. 7C).
Dominant active src kinase potentiates the coactivation function of SRC-1 on ERdependent gene transcription. The experiments measuring the effect of src kinase on
GAL-SRC-1 activity were performed in the absence of ER (as shown in Fig. 7). It was of
interest to determine whether the src potentiation of SRC-1 activity contributed to the
overall potentiation of ER-dependent transcription by src. To assess the SRC-1
contribution, SRC-1, ER, and ERE2e1b-luciferase were cotransfected in HeLa cells and
luciferase activity was measured. To specifically assess the contribution of SRC-1, the
ER/S167A mutant, and not wt ER, was used since dominant active src does not potentiate
the activity of this mutant in HeLa cells (see Fig. 3B). Therefore, any potentiation by
dominant active src would be the result of SRC-1 coactivation and could not be attributed
to ER. Although estradiol induced ER/S167A-dependent luciferase expression 2-3 fold
relative to vehicle (Fig. 8A, lane 2), dominant active src kinase failed to potentiate the
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estradiol-stimulated luciferase expression (Fig 8A, lane 4). HeLa cells express low levels
of endogenous SRC-1 when compared to Ishikawa cells (data not shown). It was
therefore likely that restoring SRC-1 levels in HeLa cells comparable to levels detected in
Ishikawa cells might uncover a src kinase contribution to ER/S167A activity that
occurred specifically through SRC-1. High efficiency transfection in HeLa cells resulted
in SRC-1 levels comparable to levels detected in Ishikawa cells (data not shown). Basal
level luciferase activity was increased two fold over vector-transfected cells and SRC-1
coactivated estradiol-dependent reporter activation 5-6 fold over control (Fig. 8A lanes 56). Only under conditions where SRC-1 was expressed did dominant active src potentiate
reporter activation under both basal conditions (10-12 fold over control) and estradiol
treatment (20 fold over control) (Fig. 8, lanes 7 and 8). These data confirm that src kinase
elevation of SRC-1 activity contributes to the overall potentiation of ER-dependent gene
transcription by specifically enhancing SRC-1 coactivation of ER.
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Discussion
A major concern with tamoxifen therapy for breast cancer is acquired resistance
as well as estrogen-like agonist action in the endometrium resulting in an increased risk
for development of endometrial cancer (35;36;54;55). Tamoxifen resistance and
tamoxifen agonist activity may result from the altered activity of cellular kinases that
change the phosphorylation status of ER and coregulators. This study describes a
mechanistic role of the non-receptor tyrosine kinase, src, in promoting the agonist action
of tamoxifen in the endometrium. In endometrial-derived Ishikawa cells, tamoxifendependent activation of reporter gene (ERE2e1b-luciferase) and endogenous gene (pS2,
c-myc) transcription was inhibited or activated by using agents that reduce or elevate src
kinase activity, respectively. The data demonstrated that src kinase modulates ERdependent transcription by altering the phosphorylation of serines 118 and 167 of ER.
Phosphorylation at serine 167 occurred via src-induced activation of the PI3K/AKT
pathway, that in turn regulated promoter interaction at both consensus and nonconsesus
EREs. Phosphorylation at serine 118 had no effect on ER promoter interaction. To our
knowledge, this is the first report of the connection between activated src kinase and
serine 167 phosphorylation of ER. This phosphorylation underlies a major mechanism for
src kinase regulation of ER action by modulating ER interaction with endogenous
promoters. Src kinase also elevated SRC-1 activity that was sufficient to enhance
coactivation of the ER. Taken together, this report finds that src kinase potentiation of
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tamoxifen agonist action results from multiple phosphorylation and activity changes in
receptor and coactivators rather than a single phosphorylation event in one protein.
When serine 118 or serine 167 was mutated to alanine, the transcriptional activity
of ER was severely abrogated and no src kinase-mediated potentiation of estradiol action
was detected. However when these same ER mutants were assayed for promoter
interaction by ChIP, only the serine 167 mutant exhibited a decrease in promoter
interaction. These results indicate that although dephosphorylation at either site results in
decreased ER-dependent transcription, the mechanisms through which each site regulates
transcription are quite different. Previous reports and the present study have shown that
phosphorylation of serine 167 promotes DNA binding (17-19), whereas serine 118 may
have a role in coactivator binding. Dutertre et al. (56) have shown that interaction of ER
with SRC-1 or CBP is decreased when serines 104/106/118 are mutated to alanine. In
addition Endoh et al. (57) has shown that phosphorylation at serine 118 increases the
interaction of ER with p68 RNA helicase providing direct evidence for serine 118 in
coactivator binding. Thus, src kinase may affect two distinct modes of ER action;
promoter recruitment and coactivator recruitment.
There is increasing evidence that bidirectional crosstalk between ER and cellular
kinase pathways may lead to tamoxifen resistance (58;59). We found an increased
expression of activated src in tamoxifen resistant endometrial cell lines when compared
to tamoxifen sensitive breast cancer cell lines (Fig 1A). Furthermore, in HeLa cells in
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which tamoxifen is an antagonist (60;61), increasing src activity promoted tamoxifen
agonist action on gene transcription to a similar extent as estrogen treatment
(supplemental data Fig. S-2). Although c-myc and pS2 genes where not induced by
tamoxifen, transfection with a dominant active src promoted tamoxifen agonist action
above that of the dominant active src alone. By comparison, incubation with src kinase
inhibitor SU6656 further potentiated the antiestrogen effect of tamoxifen by reducing
basal gene transcription. These findings suggest that src kinase may play an important
role in the development of tamoxifen resistance.
In addition to receptor phosphorylation, src kinase impacts coactivator activity
and increases SRC-1 coactivation of ER-dependent gene transcription (Fig. 7 and 8). In
Ishikawa cells, src kinase specifically enhanced SRC-1 and CBP activity but not the
activities of SRC-2 (TIF-2/GRIP-1), SRC-3 (AIB-1/RAC-3/ACTR), and PCAF (Fig. 7A
and B). Src kinase selective modulation of SRC-1 activity is interesting in light of a
recent report by Shang et al. (33) indicating that elevated SRC-1 in endometrial cancer
cell lines was correlated with tamoxifen agonist activity when compared to breast cancer
cell lines that express lower levels of SRC-1 and exhibit tamoxifen antagonist action.
The authors further demonstrated that SRC-1, but not SRC-2 or SRC-3, was important
for the tamoxifen agonist activity in Ishikawa cells. This report by Shang et al (33) and
the present study suggest that SRC-1 is a key protein involved in tamoxifen agonist
action in endometrium not only through elevated expression levels, but also as a selective
target of src kinase pathways. These results, taken together with our recent finding of
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elevated src kinase activity in endometrium (44) suggest that SRC-1 may be a
preferential substrate of src kinase in the endometrium possibly because of higher SRC-1
expression in this tissue.
CBP activity was also elevated by src kinase in a cell specific manner. In
Ishikawa cells, src kinase increased CBP activity (Fig 7A and B) whereas in HEC1A
endometrial adenocarcinoma cells, CBP activity was not altered by src kinase (Fig 7C).
A number of proteins have been reported as substrates of src kinase (62;63). Substrate
specificity by src could simply be related to the relative expression levels of these
substrates in a given tissue/cell line. It is likely that src kinase targets a distinct repertoire
of coregulators in different tissues and src effects on these coregulators could influence
estrogen and tamoxifen signaling. This may also explain the differences between the
present study examining coactivator activity in endometrial-derived cells and a previous
report by Feng et al (42) in which it was reported that src kinase increased the activities
of SRC-2 (TIF-2/GRIP-1) and CBP in ER-negative HeLa cells.
Although src kinase increased the activity of SRC-1 and enhanced its coactivation
function for the ER, no single SRC-1 phosphorylation site was responsible for the src
potentiation of SRC-1 activity. Several possibilities may explain these results. Since all
seven SRC-1 phosphorylation sites contain consensus sequences for serine/threonineproline directed kinases (26) it is possible that only one or few kinases contribute to
steady state SRC-1 phosphorylation. In addition, six of seven SRC-1 sites can be
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phosphorylated in-vitro by ERK-2 (26) a known downstream effector of src kinase (64).
In this regard, it is likely that src kinase increases phosphorylation at multiple SRC-1
sites and that no single phosphorylation event is sufficient to reproduce the src kinase
potentiation of ER-dependent gene transcription. Another possibility is that activation of
src kinase mediates a dephosphorylation event on SRC-1 that is required for SRC-1
potentiation of tamoxifen agonist action. Finally, a different protein in the SRC-1
complex may be a direct or indirect substrate of src kinase. These possibilities are
currently being assessed.
In summary, this report has demonstrated that multiple proteins and
phosphorylation sites are substrates of src kinase during potentiation of tamoxifen agonist
activity in endometrial-derived cells. The major mechanism by which src potentiates
tamoxifen agonist action is likely through phosphorylation at serine 167 that stabilized
interaction of ER with promoters of ER-dependent genes. Phosphorylation at ER serine
118 and phosphorylation of SRC-1 and possibly CBP also contribute to src potentiation
of ER-dependent gene transcription (Fig. 9A). In the absence of src kinase activation,
serine 167 phosphorylation is decreased resulting in reduced ER promoter interaction.
Dephosphorylation at serine 118 may lead to loss of other downstream transcriptional
effects. Inhibition of src kinase also decreased the intrinsic activities of SRC-1 and CBP
further contributing to the reduction in ER-dependent gene transcription (Fig. 9B).
Future work will focus on identifying the ER and coregulator phosphorylation
fingerprints associated with tamoxifen agonist/antagonist effects on specific genes.
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These studies may provide the mechanistic groundwork for rational design of preclinical
therapeutic strategies designed to inhibit src kinase to circumvent tamoxifen resistant
breast cancer and relieve the deleterious estrogen-like side effects of tamoxifen and other
SERMs in non-target tissues.
101
Materials and Methods
Cell lines: Ishikawa and HEC1A were grown at 37°C with 5% CO2 in Dulbecco’s
modified Eagle’s medium (DMEM) (Cellgro) supplemented with 5% fetal bovine serum
(FBS) (Gibco) and 1% penicillin-streptomycin (Gibco). MCF-7 were grown at 37°C
with 5% CO2 in DMEM supplemented with 10% FBS, 4 mM of glutamine (Gibco), and
1% penicillin-streptomycin. HeLa cells were grown at 37°C with 5% CO2 in DMEM
supplemented with 10% FBS, 4 mM of glutamine and 1% penicillin-streptomycin. T47D
were grown at 37°C with 5% CO2 in RPMI 1640 (Gibco) supplemented with 10% FBS,
1% penicillin-streptomycin, and 5µg/mL of insulin (Sigma).
Plasmids: Mutagenesis of ER/S104A, ER/S106A, ER/S118A, and ER/S167A was
performed using the Stratagene Quick Change Kit. ER sequence and mutations were
confirmed by DNA sequencing. ER-ABCD and ER-CDEF domain mutants were
constructed by PCR amplifying nucleotides, which correspond to amino acids 1-302 for
ABCD and 180-593 for CDEF. The resulting amplicons were subcloned into pCR3.1
vector. pBABE dominant active src (src Y527F) was a gift from Dr. Kavita Shah
(Genomics Institute of the Novartis Research Foundation). SRC-1 and Gal-SRC-1 were
constructed as previously described (27). Gal-SRC-2 and Gal-SRC-3 were gifts from Dr.
Carolyn Smith (Baylor College of Medicine). Gal-SRC-1/S372A, Gal-SRC-1/S395A,
Gal-SRC-1/S517A, Gal-SRC-1/S569A, Gal-SRC-1/S1033A, and Gal-SRC-
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1/T1179/S1185A were constructed by digesting pCR3.1-SRC-1/S372A, -SRC-1/S395A,
-SRC-1/S517A, -SRC-1/S569A, -SRC-1/S1033A, and -SRC-1/T1179/S1185A at Acc1
sites and subcloning into Gal-SRC-1 expression vector. siRNA for c-src was constructed
by annealing the sense oligo5’GCAGACATA
GAAGAGCCAATTCAAGAGATTGGCTC TTCTATGTCTGCTTTTTT 3’ to the
antisense oligo5’ AATTAAAAAAGCAGACATAGAAGAGCCAATCTCTTGAA
TTGGCTCTTCTATGTCTGCGGCC 3’ and ligating the annealed oligos into the
pSilencer 1.0 U6 vector (Ambion).
Luciferase assay: Ishikawa, HEC1A or HeLa cells were plated in 6-well plates (2×105
cells/well) and cultured in phenol red free DMEM containing 2% FBS that was charcoal
stripped to remove endogenous steroids. The cells were transfected with expression
vector as indicated in the figure legends using Fugene transfection reagent (Roche). 24 h
posttransfection, the cells were incubated with vehicle, estradiol (10-8 M) (Sigma), or 4hydroxytamoxifen (10-7 M) (Sigma) for 24 h. In experiments in which 1µM wortmannin
(Upstate Biotechnology), 1µM SU6656 (Calbiochem) or 10µM AKT inhibitor III
(Calbiochem) was used, the cells were incubated for 2 h with the inhibitors followed by
incubation with estradiol (10-8 M) and 4-hydroxytamoxifen (10-7 M) for 24 h. Luciferase
expression was measured and normalized as described previously (65).
Real-Time RT-PCR: Ishikawa and HeLa cells were plated in 100mm plates (2×106
cells/plates) in 2% stripped FBS in DMEM. Ishikawa cells were maintained in the
103
stripped media for 3 days until 90% confluency. For HeLa cells, 24h post-plating the
cells were transfected with expression vector for hER (500ng/plate), and dominant active
src (3µg/plate) or empty vector (3µg/plate) and maintained in 2% stripped FBS in
DMEM for an additional 48h. The cells were incubated with vehicle, estradiol (10-8 M),
or 4-hydroxytamoxifen (10-7 M) for 6h. In experiments in which 1µM SU6656 was used,
the cells were incubated for 2 h with the inhibitor followed by incubation with estradiol
(10-8 M) and 4-hydroxytamoxifen (10-7 M) for 6 h. Total mRNA was extracted from the
cell pellet, reverse transcribed and gene expression was measured by real-time RT-PCR
as described previously (40). Briefly, total RNA was extracted using Trizol (Invitrogen)
according to the manufacturer instructions. 200ng of mRNA was reverse transcribed
using Taqman Kit reagents (Applied Biosystems). Primers and probes for GAPDH were
designed and manufactured by Applied Biosystems and primers and probes for c-myc
and pS2 were synthesized by Integrated DNA Technologies, Inc.
C-myc:
FWD-5’-CGTCTCCACACATCAGCACAA -3’
REV-5’-TGTTGGCAGCAGGATAGTCCTT -3’
Probe-5’-56FAM/ACGCAGCGCCTCCCTCCACTC/3BHQ-1/-3’
pS2:
FWD-5’-CGTGAAAGACAGAATTGTGGTTTT-3’
REV-5’- CGTCGAAACAGCAGCCCTTA-3’
104
Probe-5’-/56FAM/ TGTCACGCCCTCCCAGTGTGCA/3BHQ-1/-3’
Fwd primer, rev primer, and probe for c-myc, pS2, or GAPDH and Taqman Universal
Master Mix (Applied Biosystems) were added to 200ng of cDNA. Real-time RT-PCR
was performed using a GeneAmp 5700 Sequence Detection System (Perkin Elmer).
Chromatin Immunoprecipitation (ChIP) Assay: Ishikawa and HeLa cells were plated
in 100mm plates (2×106cells/plates) in 2% stripped FBS in DMEM. Ishikawa cells were
maintained in the stripped media for 3 days until 90% confluent. Ishikawa cells were
incubated with vehicle, estradiol (10-8 M), or 4-hydroxytamoxifen (10-7 M) for 2.5 h. In
experiments in which 1µM wortmannin or 1µM SU6656 was used, the cells were
incubated for 2 h with the inhibitor followed by incubation with estradiol (10-8 M) or 4hydroxytamoxifen (10-7 M) for 2.5 h. Ishikawa cells were washed twice with ice cold
phosphate buffered saline (PBS) and fixed with formaldehyde (1% final concentration)
for 10 min at room temperature. The cells were washed twice with ice cold PBS and then
collected in 100mM Tris-HCl (pH 9.0) and 10mM dithiothreitol (DTT). Ishikawa cells
were incubated at 30°C for 15 min. Cells were rinsed twice with ice cold PBS, and a
nuclear extraction was performed by swelling the cells on ice in 20 volumes of nuclei
extraction buffer (5 mM piperazine-N, N’-bis 2-ethanesulfonic acid (pH 8.0) (PIPES), 85
mM KCl, 0.5% Nonidet P-40, and protease inhibitor cocktail (Sigma)) for 30 min. Nuclei
were collected by centrifugation at 250 × g for 10 min. The nuclear pellet was washed
once with nuclei extraction buffer and resuspended in 200 µl of ChIP lysis buffer (1%
105
SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1, and protease inhibitor cocktail). For
HeLa cells, 24h post-plating the cells were transfected with expression vector for either
ER or ER phosphorylation mutants (500ng/plate), and either dominant active src
(3µg/plate) or empty vector (3µg/plate) and maintained in 2% stripped FBS in DMEM
for an additional 48h. The cells were incubated with vehicle, estradiol (10-8 M), or 4hydroxytamoxifen (10-7 M) for 2.5h. In experiments in which 1µM SU6656 was used,
the cells were incubated for 2 h with the inhibitor followed by incubation with estradiol
(10-8 M) or 4-hydroxytamoxifen (10-7 M) for 2.5 h. HeLa cells were washed twice with
ice cold PBS and fixed with formaldehyde (1% final concentration) for 10 min at room
temperature. The cells were washed twice with ice cold PBS. The cells were then
collected in 100mM Tris-HCl (pH 9.0) and 10mM DTT and incubated at 30°C for 15
min. Following the 15 min incubation, the cells were rinsed twice with ice cold PBS, and
lysed in ChIP lysis buffer. The soluble chromatin for Ishikawa and HeLa cells were
sonicated three times for 10 sec at setting 4 (Fisher Sonic Dismembrator, Model 550) and
centrifuged at 20,000 × g for 15 min to remove cellular debris. The supernatant was
diluted 10 fold in ChIP dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl,
20 mM Tris-HCl, pH 8.1 and protease cocktail inhibitor) followed by preclearing with
10µL of normal mouse IgG (Santa Cruz) and 60µL of a 50% gel slurry of protein Asepharose beads (Amersham Biosciences). The beads were prepared by reswelling in
PBS and then blocking overnight in 0.1% bovine serum albumin (BSA) in PBS. To 1.5
mL of packed beads, 600µg of sonicated salmon sperm DNA and 1.5 mg of BSA was
added to a final volume of 3mL in 10mM Tris-HCl (pH 8.0), 1mM EDTA, and .05%
106
sodium azide giving a final 50% gel slurry. After preclearing, the beads were microfuged
for 1 min at 4000 × g and 20µL of supernatant was set aside for input. 10uL of anti-ERα
D-12 antibody or SP1 antibody (Santa Cruz) was added to the supernatant and the
samples were immunoprecipitated overnight at 4°C. Precipitates were washed for 5 min
each wash first in low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM
Tris-HCl, pH 8.1, 150 mM NaCl), then in high salt buffer (0.1% SDS, 1% Triton X-100,
2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl), and finally in LiCl buffer (0.25
M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1).
Precipitates were then washed 3 × 5 min with TE buffer (10mM Tris-HCl (pH 8.0), 1mM
EDTA).
Precipitated chromatin complexes were removed from the beads and
formaldehyde cross-linking was reversed by incubation for 6h at 65°C in 100 µL of 1%
SDS, 0.1 M NaHCO3 with vortexing several times throughout the incubation period.
DNA was purified with a QIAquick Spin Kit (Qiagen) according to manufacturer’s
instructions. 10µL of extracted DNA was amplified using Accuprime taq polymerase
(Invitrogen), and visualized by ethidium bromide stained 1.5% agarose gels.
c-myc nonconsesus ERE region:
FWD-5’- AGGCGCGCGTAGTTAATTCAT-3’
REV-5’- CGCCCTCTGCTTTGGGA-3’
c-myc ERE region:
FWD-5’- GATCCTCTCTCGCTAATCTCC-3’
107
REV-5’- CGCTGAAATTACTACAGCGAG-3’
pS2 ERE region:
FWD-5’-GGCCATCTCTCACTATGAATCACTTCT -3’
REV-5’- GGCAGGCTCTGTTTGCTTAAAGAGCG-3’
HTERT SP1 region:
FWD-5'-TGCCCCTTCACCTTCCAG-3'
REV-5'-CAGCGCTGCCTGAAACTC-3
Quantitative ChIP Assays: Chromatin was prepared as described above. 10µL of
extracted DNA was used in a real-time RT-PCR reaction for the detection of c-myc and
pS2 promoters.
c-myc nonconsesus ERE region:
FWD-5’- GGTAGGCGCGCGTAGTTAAT -3’
REV-5’- GGGCAGCCGAGCACTCT -3’
Probe-5’-56FAM/ ATGCGGCTCTCTTACTCTGTTTACATCCTAGAGC /3BHQ-1/-3’
c-myc ERE region:
FWD-5’- GGCTTGGCGGGAAAAAGA -3’
REV-5’- GGGCAGCCGAGCACTCT -3’
108
Probe-5’-56FAM/ATGCGGCTCTCTTACTCTGTTTACATCCTAGAGC/3BHQ-1/-3’
pS2 ERE region:
FWD-5’-TCAGATCCCTCAGCCAAGATG -3’
REV-5’- TGGTCAAGCTACATGGAAGGATT-3’
Probe-5’-56FAM/CCTCACCACATGTCGTCTCTGTCT/3BHQ-1/-3’
Values were normalized to input samples.
Western Blot Analysis: Ishikawa and HeLa cells were plated and transfected as
described above. For the detection of phospho-ER and phospho-AKT, Ishikawa cells
were serum deprived for 24 hours.
Following serum deprivation, the cells were
stimulated by incubation with vehicle, estradiol (10-8 M), or 4-hydroxytamoxifen (10-7 M)
for 30 min. In experiments in which 1µM SU6656 was used, the cells were incubated for
2 h with the inhibitor followed by incubation with estradiol (10-8 M) and 4hydroxytamoxifen (10-7 M) for 30 min. In experiments in which kinase inhibitor efficacy
was assessed, Ishikawa cells were serum deprived for 24 hours.
Following serum
deprivation, cells were incubated with either vehicle, 1µM SU6656, 1µM wortmannin, or
10µM AKT inhibitor, and then stimulated by incubation with FBS (20% final
concentration) for 30 min. The cells were lysed in high salt extraction buffer (10 mM
Tris-HCl pH 8, 0.4 M NaCl, 2 mM EDTA, 2 mM EGTA, 50 mM potassium phosphate,
50 mM sodium fluoride, 10 mM β-mercaptoethanol, 0.1% triton X-100, 0.2% protease
inhibitor cocktail, and 0.1% PMSF). For detection of phospho-AKT and SRC-1 in
109
Ishikawa cells, ER and SRC-1 in HeLa cells and active src in MCF-7, T47D, HEC1A,
and Ishikawa cells, whole cell lysate was used by lysing the cells in high salt extraction
buffer. 50 µg of whole cell or nuclear lysate was prepared for Western blotting as
previously described (44). Briefly, proteins were separated by electrophoresis on a SDSPAGE gel. The gel was transferred to nitrocellulose membrane, and the membrane was
blocked in 5% nonfat milk in tris-buffered saline/Tween 20 (TBST) (10 mM tris, pH 8,
150 mM NaCl, 0.1% Tween-20) for 1h at room temperature, followed by washes with 1x
TBST.
The membranes were incubated with primary antibodies against ER
(Novacastra), phospho-ER 104/106, phospho-ER 118, phospho-ER 167, non-phospho527 src, phospho-AKT, AKT, (Cell Signaling Technology) src (Upstate Biotechnology),
and SRC-1 and β-actin (Santa Cruz).
Following the primary antibody incubation,
horseradish peroxidase conjugated secondary antibody (Vector) was incubated for 1h at
room temperature in 5% nonfat milk in TBST. The signal was developed by addition of
enhanced chemiluminescence (ECL) solution. The membranes were exposed to Kodak
XOMAT film, and/or the signal from the membrane was quantitated using a Kodak
Digital Science 1D image analysis software station 440 system.
Validation
of
siRNA-src:
Ishikawa
cells
(2×106cells/plates) in 5% FBS in DMEM.
were
plated
in
100mm
plates
Ishikawa cells were cultured until cells
reached 90% confluency. Ishikawa were transfected with either siRNA-src or vector
(20µg/plate) using Lipofectamine 2000 (Invitrogen) according to manufacturer’s
110
instructions. 72 hours posttransfection the cells were harvested for Western Blot analysis
measuring src levels as described above.
Data Analysis: All data are expressed as ± S.D. P values were calculated using Anova
Dunnett’s T Test and Independent T Test. P<.05 was considered significant.
Acknowledgements: This work was supported in part by National Institutes of Health
Grant RO1 DK06832 (to B.G.R.) and by a Department of Defense Breast Cancer
Research Program Idea Award (DAMD17-02-1-0531) and Career Development Award
(DAMD17-02-1-0530) (to B.G.R). Y.K.S. was supported by a Predoctoral Dissertation
Award (DISS0100539) from the Susan G. Komen Breast Cancer Foundation.
111
Reference List
1. Klinge CM 2001 Estrogen receptor interaction with estrogen response elements.
Nucleic Acids Res 29:2905-2919
2. Tsai MJ, O'Malley BW 1994 Molecular mechanisms of action of steroid/thyroid
receptor superfamily members. Annu Rev Biochem 63:451-86.:451-486
3. Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Lin SC, Heyman RA,
Rose DW, Glass CK, Rosenfeld MG 1996 A CBP integrator complex mediates
transcriptional activation and AP-1 inhibition by nuclear receptors. Cell 85:403-414
4. Onate SA, Tsai SY, Tsai MJ, O'Malley BW 1995 Sequence and characterization of
a coactivator for the steroid hormone receptor superfamily. Science 270:1354-1357
5. Hong H, Kohli K, Trivedi A, Johnson DL, Stallcup MR 1996 GRIP1, a novel
mouse protein that serves as a transcriptional coactivator in yeast for the hormone
binding domains of steroid receptors. Proc Natl Acad Sci U S A 93:4948-4952
6. Voegel JJ, Heine MJ, Zechel C, Chambon P, Gronemeyer H 1996 TIF2, a 160 kDa
transcriptional mediator for the ligand-dependent activation function AF-2 of
nuclear receptors. EMBO J 15:3667-3675
7. Anzick SL, Kononen J, Walker RL, Azorsa DO, Tanner MM, Guan XY, Sauter G,
Kallioniemi OP, Trent JM, Meltzer PS 1997 AIB1, a steroid receptor coactivator
amplified in breast and ovarian cancer. Science 277:965-968
8. Chen H, Lin RJ, Schiltz RL, Chakravarti D, Nash A, Nagy L, Privalsky ML,
Nakatani Y, Evans RM 1997 Nuclear receptor coactivator ACTR is a novel histone
acetyltransferase and forms a multimeric activation complex with P/CAF and
CBP/p300. Cell 90:569-580
9. Li H, Gomes PJ, Chen JD 1997 RAC3, a steroid/nuclear receptor-associated
coactivator that is related to SRC-1 and TIF2. Proc Natl Acad Sci U S A 94:84798484
10. Takeshita A, Yen PM, Misiti S, Cardona GR, Liu Y, Chin WW 1996 Molecular
cloning and properties of a full-length putative thyroid hormone receptor
coactivator. Endocrinology 137:3594-3597
11. Torchia J, Rose DW, Inostroza J, Kamei Y, Westin S, Glass CK, Rosenfeld MG
1997 The transcriptional co-activator p/CIP binds CBP and mediates nuclearreceptor function. Nature 387:677-684
112
12. Chakravarti D, LaMorte VJ, Nelson MC, Nakajima T, Schulman IG, Juguilon H,
Montminy M, Evans RM 1996 Role of CBP/P300 in nuclear receptor signalling.
Nature 383:99-103
13. Shibata H, Spencer TE, Onate SA, Jenster G, Tsai SY, Tsai MJ, O'Malley BW 1997
Role of co-activators and co-repressors in the mechanism of steroid/thyroid receptor
action. Recent Prog Horm Res 52:141-64; discussion 164-5.:141-164
14. Le Goff P, Montano MM, Schodin DJ, Katzenellenbogen BS 1994 Phosphorylation
of the human estrogen receptor. Identification of hormone-regulated sites and
examination of their influence on transcriptional activity. J Biol Chem 269:44584466
15. Lahooti H, White R, Danielian PS, Parker MG 1994 Characterization of liganddependent phosphorylation of the estrogen receptor. Mol Endocrinol 8:182-188
16. Ali S, Metzger D, Bornert JM, Chambon P 1993 Modulation of transcriptional
activation by ligand-dependent phosphorylation of the human oestrogen receptor
A/B region. EMBO J 12:1153-1160
17. Tzeng DZ, Klinge CM 1996 Phosphorylation of purified estradiol-liganded
estrogen receptor by casein kinase II increases estrogen response element binding
but does not alter ligand stability. Biochem Biophys Res Commun 223:554-560
18. Castano E, Vorojeikina DP, Notides AC 1997 Phosphorylation of serine-167 on the
human oestrogen receptor is important for oestrogen response element binding and
transcriptional activation. Biochem J 326:149-157
19. Arnold SF, Obourn JD, Jaffe H, Notides AC 1995 Phosphorylation of the human
estrogen receptor by mitogen-activated protein kinase and casein kinase II:
consequence on DNA binding. J Steroid Biochem Mol Biol 55:163-172
20. Bunone G, Briand PA, Miksicek RJ, Picard D 1996 Activation of the unliganded
estrogen receptor by EGF involves the MAP kinase pathway and direct
phosphorylation. EMBO J 15:2174-2183
21. Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H, Masushige S,
Gotoh Y, Nishida E, Kawashima H, . 1995 Activation of the estrogen receptor
through phosphorylation by mitogen-activated protein kinase. Science 270:14911494
22. Lannigan DA 2003 Estrogen receptor phosphorylation. Steroids 68:1-9
23. Sun M, Paciga JE, Feldman RI, Yuan Z, Coppola D, Lu YY, Shelley SA, Nicosia
SV, Cheng JQ 2001 Phosphatidylinositol-3-OH Kinase (PI3K)/AKT2, activated in
113
breast cancer, regulates and is induced by estrogen receptor alpha (ERalpha) via
interaction between ERalpha and PI3K. Cancer Res 61:5985-5991
24. Martin MB, Franke TF, Stoica GE, Chambon P, Katzenellenbogen BS, Stoica BA,
McLemore MS, Olivo SE, Stoica A 2000 A role for Akt in mediating the estrogenic
functions of epidermal growth factor and insulin-like growth factor I.
Endocrinology 141:4503-4511
25. Campbell RA, Bhat-Nakshatri P, Patel NM, Constantinidou D, Ali S, Nakshatri H
2001 Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor
alpha: a new model for anti-estrogen resistance. J Biol Chem 276:9817-9824
26. Rowan BG, Weigel NL, O'Malley BW 2000 Phosphorylation of steroid receptor
coactivator-1. Identification of the phosphorylation sites and phosphorylation
through the mitogen-activated protein kinase pathway. J Biol Chem 275:4475-4483
27. Rowan BG, Garrison N, Weigel NL, O'Malley BW 2000 8-Bromo-cyclic AMP
induces phosphorylation of two sites in SRC-1 that facilitate ligand-independent
activation of the chicken progesterone receptor and are critical for functional
cooperation between SRC-1 and CREB binding protein. Mol Cell Biol 20:87208730
28. Font dM, Brown M 2000 AIB1 is a conduit for kinase-mediated growth factor
signaling to the estrogen receptor. Mol Cell Biol 20:5041-5047
29. Lopez GN, Turck CW, Schaufele F, Stallcup MR, Kushner PJ 2001 Growth factors
signal to steroid receptors through mitogen-activated protein kinase regulation of
p160 coactivator activity. J Biol Chem 276:22177-22182
30. Wagner BL, Norris JD, Knotts TA, Weigel NL, McDonnell DP 1998 The nuclear
corepressors NCoR and SMRT are key regulators of both ligand- and 8-bromocyclic AMP-dependent transcriptional activity of the human progesterone receptor.
Mol Cell Biol 18:1369-1378
31. Hong SH, Privalsky ML 2000 The SMRT corepressor is regulated by a MEK-1
kinase pathway: inhibition of corepressor function is associated with SMRT
phosphorylation and nuclear export. Mol Cell Biol 20:6612-6625
32. Lavinsky RM, Jepsen K, Heinzel T, Torchia J, Mullen TM, Schiff R, Del Rio AL,
Ricote M, Ngo S, Gemsch J, Hilsenbeck SG, Osborne CK, Glass CK, Rosenfeld
MG, Rose DW 1998 Diverse signaling pathways modulate nuclear receptor
recruitment of N-CoR and SMRT complexes. Proc Natl Acad Sci U S A 95:29202925
114
33. Shang Y, Brown M 2002 Molecular determinants for the tissue specificity of
SERMs. Science 295:2465-2468
34. Jepsen K, Hermanson O, Onami TM, Gleiberman AS, Lunyak V, McEvilly RJ,
Kurokawa R, Kumar V, Liu F, Seto E, Hedrick SM, Mandel G, Glass CK, Rose
DW, Rosenfeld MG 2000 Combinatorial roles of the nuclear receptor corepressor in
transcription and development. Cell 102:753-763
35. Harper MJ, Walpole AL 1967 A new derivative of triphenylethylene: effect on
implantation and mode of action in rats. J Reprod Fertil 13:101-119
36. Terenius L 1971 Structure-activity relationships of anti-oestrogens with regard to
interaction with 17-beta-oestradiol in the mouse uterus and vagina. Acta Endocrinol
(Copenh) 66:431-447
37. Sakamoto T, Eguchi H, Omoto Y, Ayabe T, Mori H, Hayashi S 2002 Estrogen
receptor-mediated effects of tamoxifen on human endometrial cancer cells. Mol
Cell Endocrinol 192:93-104
38. Kleinman D, Karas M, Danilenko M, Arbell A, Roberts CT, LeRoith D, Levy J,
Sharoni Y 1996 Stimulation of endometrial cancer cell growth by tamoxifen is
associated with increased insulin-like growth factor (IGF)-I induced tyrosine
phosphorylation and reduction in IGF binding proteins. Endocrinology 137:10891095
39. Anzai Y, Holinka CF, Kuramoto H, Gurpide E 1989 Stimulatory effects of 4hydroxytamoxifen on proliferation of human endometrial adenocarcinoma cells
(Ishikawa line). Cancer Res 49:2362-2365
40. Kershah SM, Desouki MM, Koterba KL, Rowan BG 2004 Expression of estrogen
receptor coregulators in normal and malignant human endometrium. Gynecol Oncol
92:304-313
41. Lee H, Jiang F, Wang Q, Nicosia SV, Yang J, Su B, Bai W 2000 MEKK1
activation of human estrogen receptor alpha and stimulation of the agonistic activity
of 4-hydroxytamoxifen in endometrial and ovarian cancer cells. Mol Endocrinol
14:1882-1896
42. Feng W, Webb P, Nguyen P, Liu X, Li J, Karin M, Kushner PJ 2001 Potentiation of
estrogen receptor activation function 1 (AF-1) by Src/JNK through a serine 118independent pathway. Mol Endocrinol 15:32-45
43. Reissig D, Clement J, Sanger J, Berndt A, Kosmehl H, Bohmer FD 2001 Elevated
activity and expression of Src-family kinases in human breast carcinoma tissue
versus matched non-tumor tissue. J Cancer Res Clin Oncol 127:226-230
115
44. Desouki MM, Rowan BG 2004 SRC kinase and mitogen-activated protein kinases
in the progression from normal to malignant endometrium. Clin Cancer Res 10:546555
45. Irby RB, Yeatman TJ 2000 Role of Src expression and activation in human cancer.
Oncogene 20;19:5636-5642
46. Thomas SM, Brugge JS 1997 Cellular functions regulated by Src family kinases.
Annu Rev Cell Dev Biol 13:513-609.:513-609
47. Abram CL, Courtneidge SA 2000 Src family tyrosine kinases and growth factor
signaling. Exp Cell Res 254:1-13
48. Krasilnikov MA 2000 Phosphatidylinositol-3 kinase dependent pathways: the role
in control of cell growth, survival, and malignant transformation. Biochemistry
(Mosc ) 65:59-67
49. Dubik D, Shiu RP 1992 Mechanism of estrogen activation of c-myc oncogene
expression. Oncogene 7:1587-1594
50. Stack G, Kumar V, Green S, Ponglikitmongkol M, Berry M, Rio MC, Nunez AM,
Roberts M, Koehl C, Bellocq P, . 1988 Structure and function of the pS2 gene and
estrogen receptor in human breast cancer cells. Cancer Treat Res 40:185-206.:185206
51. Lee H, Jiang F, Wang Q, Nicosia SV, Yang J, Su B, Bai W 2000 MEKK1
activation of human estrogen receptor alpha and stimulation of the agonistic activity
of 4-hydroxytamoxifen in endometrial and ovarian cancer cells. Mol Endocrinol
14:1882-1896
52. Liu XF, Bagchi MK 2004 Recruitment of Distinct Chromatin-modifying
Complexes by Tamoxifen-complexed Estrogen Receptor at Natural Target Gene
Promoters in Vivo. J Biol Chem 279:15050-15058
53. Won J, Yim J, Kim TK 2002 Sp1 and Sp3 recruit histone deacetylase to repress
transcription of human telomerase reverse transcriptase (hTERT) promoter in
normal human somatic cells. J Biol Chem 277:38230-38238
54. Fornander T, Rutqvist LE, Cedermark B, Glas U, Mattsson A, Silfversward C,
Skoog L, Somell A, Theve T, Wilking N, . 1989 Adjuvant tamoxifen in early breast
cancer: occurrence of new primary cancers. Lancet 1:117-120
55. Fisher B, Costantino JP, Redmond CK, Fisher ER, Wickerham DL, Cronin WM
1994 Endometrial cancer in tamoxifen-treated breast cancer patients: findings from
116
the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-14. J Natl
Cancer Inst 86:527-537
56. Dutertre M, Smith CL 2003 Ligand-independent interactions of p160/steroid
receptor coactivators and CREB-binding protein (CBP) with estrogen receptoralpha: regulation by phosphorylation sites in the A/B region depends on other
receptor domains. Mol Endocrinol 17:1296-1314
57. Endoh H, Maruyama K, Masuhiro Y, Kobayashi Y, Goto M, Tai H, Yanagisawa J,
Metzger D, Hashimoto S, Kato S 1999 Purification and identification of p68 RNA
helicase acting as a transcriptional coactivator specific for the activation function 1
of human estrogen receptor alpha. Mol Cell Biol 19:5363-5372
58. Nicholson RI, McClelland RA, Robertson JF, Gee JM 1999 Involvement of steroid
hormone and growth factor cross-talk in endocrine response in breast cancer.
Endocr Relat Cancer 6:373-387
59. Schiff R, Massarweh S, Shou J, Osborne CK 2003 Breast cancer endocrine
resistance: how growth factor signaling and estrogen receptor coregulators
modulate response. Clin Cancer Res 9:447S-454S
60. Smith CL, Conneely OM, O'Malley BW 1993 Modulation of the ligandindependent activation of the human estrogen receptor by hormone and
antihormone. Proc Natl Acad Sci U S A 90:6120-6124
61. Smith CL, Nawaz Z, O'Malley BW 1997 Coactivator and corepressor regulation of
the agonist/antagonist activity of the mixed antiestrogen, 4-hydroxytamoxifen. Mol
Endocrinol 11:657-666
62. Courtneidge SA 2003 Isolation of novel Src substrates. Biochem Soc Trans 31:2528
63. Brown MT, Cooper JA 1996 Regulation, substrates and functions of src. Biochim
Biophys Acta 1287:121-149
64. Schlaepfer DD, Jones KC, Hunter T 1998 Multiple Grb2-mediated integrinstimulated signaling pathways to ERK2/mitogen-activated protein kinase:
summation of both c-Src- and focal adhesion kinase-initiated tyrosine
phosphorylation events. Mol Cell Biol 18:2571-2585
65. Coleman KM, Dutertre M, El Gharbawy A, Rowan BG, Weigel NL, Smith CL
2003 Mechanistic differences in the activation of estrogen receptor-alpha (ER
alpha)- and ER beta-dependent gene expression by cAMP signaling pathway(s). J
Biol Chem 278:12834-12845
117
Figure Legends
Fig. 1.
Src kinase regulates ER-dependent transcription of ERE2e1b-luciferase in
Ishikawa cells. (A) Increased expression of activated src kinase in endometrial- versus
breast-derived cell lines. MCF-7, T47D, HEC1A and Ishikawa cells (2 x 106 cells/plate)
were plated in 2% charcoal stripped serum for 3 days. Protein extract was prepared from
the cells and used for Western blot analysis to measure levels of activated src kinase as
described in the Material and Methods. Quantitation of the Western blot signal was
normalized to the Western blot signal for total src and β-actin levels (right panel). Each
bar represents the mean value ± S.D. (*) = P<.05 compared to MCF-7 and T47D cells. (B
and C) Ishikawa cells (2 x 105 cells/well) were transfected with ERE2e1b-luciferase
reporter (0.5µg/well). 24 hours posttransfection cells were incubated with (A) 1µM
SU6656, a specific src kinase inhibitor, for 2 h or (B) cells were cotransfected with
dominant active src kinase (Dom) (0.5µg/well) or empty vector pBABE (0.5µg/well).
Following incubation with inhibitor or 24 hours posttransfection, cells were incubated
with 10-8 M estradiol (E2) or 10-7 M 4-hydroxytamoxifen (Tam) for 24 hours. Standard
luciferase assays were performed on cell extracts in triplicate as described in the
Materials and Methods. Each bar represents the mean value ± S.D. (*) = P<.05 compared
to estradiol or tamoxifen incubated samples alone. (†) = P<.05 compared to untreated
(Con). (D) Western blot analysis of src kinase levels following transfection of a siRNA
specific for src kinase (siRNA-src). The siRNA-src was constructed as described in the
Material and Methods. Ishikawa cells (2 x 106 cells/plate) were transfected with siRNA-
118
src (20µg/Plate). 72 h posttransfection, protein extract was prepared from Ishikawa cells
and used for Western blot analysis to measure expression levels of src kinase as described
in the Material and Methods. Quantitation of the Western blot signal was normalized to
β-actin (right panel). Each bar represents the mean value ± S.D. (*) = P<. 05. (E)
siRNA-src decreases estradiol and 4-hydroxytamoxifen activation of ERE2e1b-luciferase
reporter. Ishikawa cells were plated as described above and cotransfected with ERE2e1bluciferase reporter (0.5µg/well) and siRNA-src (0.5µg/well) or empty vector
(0.5µg/well). 72 hours posttransfection, cells were incubated with 10-8 M estradiol (E2)
or 10-7 M 4-hydroxytamoxifen (Tam) for 24 hours. Standard luciferase assays were
performed on cell extracts in triplicate as described in the Materials and Methods. Each
bar represents the mean value ± S.D. (*) = P<.05 compared to estradiol or tamoxifen
incubated samples alone.
(†) = P<.05 compared to untreated (Con) samples.
(F)
SU6656, dominant active src, or siRNA-src had no effect on a constitutively active RSVluciferase reporter. Ishikawa cells were plated as described above and transfected with a
RSV-luciferase reporter (0.5µg/well) and incubated with 1µM SU6656 or cotransfected
with either siRNA-src (0.5µg/well) or dominant active src (Dom).
24 hours
posttransfection or treatment, standard luciferase assays were performed on cell extracts
in triplicate as described in the Materials and Methods.
Fig. 2.
Activation or inhibition of src kinase regulates estradiol- and 4-
hydroxytamoxifen-dependent c-myc and pS2 gene expression. Ishikawa cells (2 x 106
cells/plate) were incubated with 1µM SU6656 for 2 h. Following incubation with the
119
inhibitor, cells were incubated with 10-8 M estradiol (E2) or 10-7 M 4-hydroxytamoxifen
(Tam) for 6 h. Expression of (A) c-myc and, (B) pS2 genes was measured by real time
RT-PCR as described in the Materials and Methods. Expression was normalized to
GAPDH and each bar represents the mean value ± S.D. (*) = P<.05 compared to
estradiol and 4-hydroxytamoxifen incubated samples alone. (†) = P<.05 compared to
untreated (Con) samples.
Fig. 3. Src kinase induces phosphorylation of serines 118 and 167 of ER. (A) HeLa cells
(2 x 106 cells/plate) were transfected with ER (0.5µg/plate). 24 h posttransfection the
HeLa were incubated with 1µM SU6656 for 2 h. Following incubation with the inhibitor
the cells were incubated with 10-8 M estradiol (E2) for 30 min. Protein extract was
prepared from HeLa cells and used for Western blot analysis to measure levels of
phosphorylated serines 104/106, 118 and 167 on ER as described in the Material and
Methods. Protein levels of total ER and β-actin were used as loading controls. (B)
Mutation of serine 118 and serine 167 to alanine on ER inhibits dominant active src
potentiation. HeLa cells (2 x 105 cells/well) were cotransfected with ERE2e1b-luciferase
reporter (0.5µg/well), either dominant active src (0.5µg/well) (Dom) or empty vector
pBABE (0.5µg/well), and either wild type ER or phosphorylation mutants ER/S104A,
ER/S106A, ER/S118A, or ER/S167A (0.05µg/well). 24 hours posttransfection cells were
incubated with 10-8 M estradiol (E2) for 24 hours. Standard luciferase assays were
performed on cell extracts in triplicate as described in the Materials and Methods. Each
bar represents the mean value ± S.D. (*) = P<.05 compared to estradiol incubated
120
samples alone.
(C) The decrease in transcriptional activity of ER phosphorylation
mutants was not due to ER expression levels. A portion of the extract used in part B
above was prepared for Western blot analysis for the detection of ER as described in the
Material and Methods.
Fig. 4. The src kinase inhibitor SU6656 disrupts ER promoter interaction measured by
ChIP. Ishikawa cells (2 x 106 cells/plate) were incubated with 1µM SU6656 for 2 h.
Following incubation with the inhibitor the cells were incubated with 10-8 M estradiol
(E2) or 10-7 M 4-hydroxytamoxifen (Tam) for 2.5 h. Chromatin was prepared and
immunoprecipitated with antibody against ER. The purified DNA was amplified by PCR
and visualized by ethidium bromide staining (left panel). Relative levels of promoter
interaction were measured by real time RT-PCR (right panel) using primers that span the
ERE region of the pS2 gene as described in Material and Methods. For quantitative ChIP
assays, relative levels of promoter interaction were normalized to input and each bar
represents the mean value ± S.D.
(*) = P<.05 compared to estradiol and 4-
hydroxytamoxifen incubated samples alone. (†) = P<.05 compared to untreated (Con)
samples.
Fig. 5. Src kinase potentiation of ER-dependent transcription and promoter interaction is
partially mediated via the PI3K/AKT pathway. (A) Ishikawa cells (2 x 106 cells/plate)
were incubated with 1µM SU6656 for 2 h. Following incubation with the inhibitor the
cells were incubated with 10-8 M estradiol (E2) or 10-7 M 4-hydroxytamoxifen (Tam) for
121
30 min. Protein extract was prepared from Ishikawa cells and used for Western blot
analysis to measure levels of activated AKT as described in the Material and Methods.
Quantitation of the Western blot signal was normalized to the Western blot signal for
total AKT and β-actin levels (right panel). Each bar represents the mean value ± S.D. (*)
= P<.05 compared to estradiol and 4-hydroxytamoxifen incubated samples alone. (†) =
P<.05 compared to untreated (Con) samples. (B) PI3K or (C) AKT inhibition blocks the
dominant active src potentiation of estradiol- and 4-hydroxytamoxifen-mediated gene
transcription. Ishikawa cells (2 x 105 cells/well) were cotransfected with ERE2e1bluciferase reporter (0.5µg/well) and either dominant active src (0.5µg/well) (Dom) or
empty vector pBABE (0.5µg/well). 24 hours posttransfection the cells were incubated
with 10-8 M estradiol (E2) or 10-7 M 4-hydroxytamoxifen (Tam). For the samples in
which 1µM wortmannin (WORT) or 10µM AKT inhibitor (AKT INH) was used, the
cells were incubated for 2 h with the inhibitor followed by incubation with 10-8 M
estradiol (E2) or 10-7 M 4-hydroxytamoxifen (Tam). Standard luciferase assays were
performed on cell extracts in triplicate as described in the Materials and Methods. Each
bar represents the mean value ± S.D.
(*) = P<.05 compared to estradiol and 4-
hydroxytamoxifen incubated samples alone. (†) = P<.05 compared to estradiol and 4hydroxytamoxifen in combination with dominant active src. (D) Wortmannin disrupts
ER promoter interaction. Ishikawa cells (2 x 106 cells/plate) were incubated with 1µM
wortmannin (WORT) for 2 h. Following incubation with the inhibitor the cells were
incubated with 10-8 M estradiol (E2) or 10-7 M 4-hydroxytamoxifen (Tam) for 2.5 h.
Chromatin was prepared and immunoprecipitated with antibody against ER. The purified
122
DNA was amplified by PCR and visualized by ethidium bromide staining (left panel) or
relative levels of promoter interaction were measured by real time RT-PCR (right panel)
using primers that span the ERE region of the pS2 gene, (as described in the Material and
Methods. For quantitative ChIP assays, relative levels of promoter interaction were
normalized to input and each bar represents the mean value ± S.D. (*) = P<.05 compared
to estradiol and 4-hydroxytamoxifen incubated samples alone. (†) = P<.05 compared to
untreated (Con) samples.
Fig. 6. Src kinase modulates promoter interaction through serine 167 phosphorylation of
ER. HeLa cells (2 x 106 cells/plates) were cotransfected with either dominant active src
(0.5µg/well) (Dom) or empty vector pBABE (3µg/well) and either wild type ER or
phosphorylation
mutants
ER/S118A
or
ER/S167A
(0.5µg/well).
24
hours
posttransfection the cells were incubated with 10-8 M estradiol (E2) for 2.5 h. Chromatin
was prepared and immunoprecipitated with antibody against ER. The purified DNA was
amplified by real time RT-PCR using primers that span the ERE region of the pS2 gene,
as described in the Material and Methods. Relative levels of promoter interaction were
normalized to input and each bar represents the mean value ± S.D. (*) = P<.05 compared
to estradiol incubated samples alone. (†) = P<.05 compared to untreated (Con) samples.
Fig. 7. Src kinase increases the activity of SRC-1 and CBP. (A and B) Ishikawa cells (2
x 105 cells/well) were cotransfected with GalRE-luciferase (0.5µg/well), dominant active
src (Dom) (0.5µg/well), or siRNA-src (0.5µg/well) and Gal-SRC-1, Gal-SRC-2, Gal-
123
SRC-3, Gal-PCAF, Gal-CBP, or GAL-DBD (0.5µg/well). 48 hours posttransfection
standard luciferase assays were performed on cell extracts in triplicate as described in the
Materials and Methods.
Each bar represents the mean value ± S.D.
(*) = P<.05
compared to GAL-DBD. (B) The samples receiving src kinase inhibitor 1µM SU6656
(INH) were transfected with GalRE-luciferase and Gal-SRC-1, Gal-SRC-2, Gal-SRC-3,
Gal-PCAF, Gal-CBP, or GAL-DBD (0.5µg/well). 24 hours posttransfection the cells
were incubated with 1µM SU6656 (INH) for 24 hours. Standard luciferase assays were
performed on cell extracts in triplicate as described in the Materials and Methods. Each
bar represents the mean value ± S.D. (*) = P<.05 compared to the GAL-DBD. (C) Src
kinase increases SRC-1 activity in HEC1A cells. HEC1A cells (2 x 105 cells/well) were
cotransfected with GalRE-luciferase (0.5µg/well), either dominant active src (Dom)
(0.5µg/well) or empty vector pBABE (0.5µg/well), either siRNA-src (0.5µg/well) or
empty vector (0.5µg/well), and either Gal-SRC-1, Gal-SRC-2, Gal-SRC-3, Gal-PCAF,
Gal-CBP, or GAL-DBD (0.5µg/well). For samples receiving the inhibitor, 24 hours
posttransfection the cells were incubated with 1µM SU6656 (INH) for 24 hours.
Standard luciferase assays were performed on cell extracts in triplicate as described in the
Materials and Methods.
Each bar represents the mean value ± S.D.
(*) = P<.05
compared to the GAL-coactivator constructs alone.
Fig. 8. Src kinase increases SRC-1 coactivation on ER-dependent gene transcription.
HeLa cells (2 x 105 cells/well) were cotransfected with ER/S167A (0.05µg/well), either
124
dominant active src (Dom) (0.5µg/well) or empty vector pBABE (0.5µg/well), and either
SRC-1 (0.5µg/well) or empty vector pCR3.1 (0.5µg/well). 24 hours posttransfection,
cells were incubated with 10-8 M estradiol (E2) for 24 h. Standard luciferase assays were
performed on cell extracts in triplicate as described in the Materials and Methods. Each
bar represents the mean value ± S.D. (*) = P<.05 compared to cells transfected with
ER/S167A, dominant active src and SRC-1. (†) = P<.05 compared to cells transfected
with ER/S167A and SRC-1.
Fig. 9. Model of src kinase potentiation of estradiol- and 4-hydroxytamoxifen-dependent
gene transcription. (A) Activation of src kinase increases ERK1/2 and AKT activity that
results in phosphorylation of ER on serines 118 and 167, respectively.
Src kinase
activation also increases the activity of coactivators SRC-1 and CBP through unknown
pathways.
(B) In the absence of src kinase activation, serine 118 and 167
phosphorylation is decreased resulting in reduced promoter interaction and transcriptional
activity for ER. Inhibition of src kinase also decreases SRC-1 and CBP activity that is
important for coactivation of estrogen- and tamoxifen-dependent gene transcription.
125
126
om
1000000
+D
2500000
m
Ta
m
+S
U6
65
6
∗
Ta
6
1500000
m
65
1000000
2000000
†
†
∗
500000
0
∗
∗
†
†
500000
0
wa
1A
ka
EC
0
hi
H
7D
F
-7
ka
wa
1A
6
Is
C
T4
M
hi
EC
Is
H
F
-7
7D
C
T4
M
Active src/total src/β-actin
β-actin
Ta
u6
Total src
m
+S
E2
H
Active src
Ta
†
om
E2
C
+D
1500000
E2
m
IN
n
2000000
E2
Fig 1
Do
n
Co
RLU
B
Co
RLU
A
∗
5
∗
4
3
2
1
0.25
D
siRNA
Vector -Src
0.2
0.15
Src
∗
0.1
β-actin
0.05
E
siR
NA
Co
-sr
c
n
0
5000000
†
4000000
†
RLU
3000000
∗
2000000
∗
†
1000000
35000000
NA
-s r
c
m
+s
iR
Ta
Ta
m
E2
+s
iR
siR
F
NA
-s r
c
E2
-sr
c
NA
Co
n
0
30000000
25000000
20000000
15000000
10000000
5000000
siR
NA
127
3
m
2
Do
-sr
c
66
56
1
SU
Fig 1
Co
n
0
4
F ig 2
128
+S
65
6
m
pS2
u6
0
6
4
65
Ta
m
+S
6
65
6
m
65
Ta
u6
u6
+S
0
Ta
U6
B 10
E2
56
E2
66
2
m
+S
E2
Su
n
C -M Y C
Ta
E2
H
n
Co
6
IN
Co
Fold Difference
Fold Difference
A
†
5
4
3
†
∗
1
∗
†
8
6
†
∗
2
∗
W
t
S1
04
A
S1
06
A
S1
18
A
S1
67
A
Po
s
C
ER wt
ER S104A
ER S106A
ER
Fig 3
129
ER S118A
E2 E
+D 2
om
Co
Do n
m
E2 E2
+D
om
2000000
Co
Do n
m
∗
E2 E
+D 2
om
Co
Do n
m
3000000
E2 E2
+D
om
Co
n
Do
m
E2 E
+D 2
om
Co
n
Do
m
RLU
E2
+
E2
SU
66
56
Co
n
A
P-ER118
P-ER167
ER
β-actin
B
5000000
∗
4000000
∗
1000000
0
ER S167A
Fig 4
130
Ta
m
+S
U
6
6
66
56
Ta
m
66
5
E2
66
5
n
Input
E2
+S
U
SU
Co
Genomic DNA
Tam+SU6656
Con 1°AB
Tam
EST
Est+SU6656
SU6656
Con
Fold Difference
pS2
16
14
12
10
8
6
4
2
0
†
∗
†
∗
Fig 6
Fig 5
10000000
131
+D
om
IN
H+
Do
m
30000000
+A
KT
20000000
IN
H
5000000
Ta
m
+S
U6
65
6
Ta
m
0.00008
Ta
m
0.00012
Ta
m
10000000
E2
E2
+S
U6
65
6
†
Ta
m
45000000
Ta
m
+W
OR
T
Ta
Ta
m
m
+D
+I
W
om
OR
T+
Do
m
ß-actin
Ta
m
Co
n
SU
66
56
Tam+SU6656
Tam
Est+SU6656
Est
SU6656
con
pAKT/AKT/b-Actin
AKT
+A
KT
60000000
†
E2
E2
+W
OR
T
E2
E2
+
+W
Do
m
OR
T+
Do
m
P-AKT
Ta
m
E2
E2
+A
KT
IN
H
E2
E2
+D
+A
om
KT
IN
H+
Do
m
IN
H+
Do
m
Do
m
W
OR
T
Do
W
m
OR
T+
Do
m
70000000
IN
H
C
AK
T
AK
T
Co
n
RLU
B
Co
n
RLU
A
†
0.0001
†
∗
0.00006
∗
0.00004
0.00002
0
40000000
35000000
25000000
†
20000000
15000000
∗
∗
0
50000000
40000000
30000000
†
∗
∗
0
Fig 5
132
+W
∗
OR
T
4
Ta
m
6
Ta
m
Input
OR
T
n
pS2
Fold Difference
16
E
E2
+W 2
OR
T
W
Co
Genomic DNA
Con 1°AB
Tam+WORT
Tam
Est+WORT
EST
WORT
Con
D
14
†
12
10
8
†
2
∗
0
0
n
ER wt
ER S118A
Fig 6
133
2
E2
E2
+D
om
Do
m
n
∗
Co
3
E2
E2
+D
om
†
Do
m
†
n
6
Co
E2
E2
+D
om
4
Do
m
Co
Fold Difference
7
∗
pS2
5
†
†
1
†
ER S167A
A
45000000
∗
40000000
35000000
RLU
30000000
∗
25000000
20000000
15000000
∗
10000000
5000000
∗
0
Gal-DBD
Gal-SRC-1
Gal-SRC-2
Gal-SRC-3
Gal-PCAF
Gal-CBP
siRNA-Src
Dom
+ + +
- - -
- - + + +
- - - - -
- - - - -
- - - - -
-
-
-
- - - -
+ + +
- - - - -
- - + + +
- - -
- - - - + + +
- - - - - - -
-
- + - +
-
- - +
- -
- - - + - - +
+ + +
- + - - +
-
-
- - - + - - +
- + - +
+
- - -
30000000
B
25000000
RLU
20000000
∗
15000000
10000000
5000000
∗
CB
CB
P+
P
SU
66
56
SR
C3
3+
SU
66
56
SR
C-
134
PC
AF PCA
F
+S
U6
65
6
SR
2+ C -2
SU
66
56
SR
C-
SR
1+ C -1
SU
66
56
SR
C-
Fig 7
Ga
l+ Ga
SU l
66
56
0
C
30000000
∗
25000000
RLU
20000000
15000000
∗
10000000
∗ ∗
5000000
∗
∗
0
Gal-DBD + + + +
Gal-SRC-1 - - - Gal-SRC-2 - - - Gal-SRC-3 - - - Gal-PCAF - - - Gal-CBP
SU6656
siRNA-Src
Dom
- - - -
- - - -
- - - -
- - - -
- - - -
+ + + +
- - - -
- - - -
- - - -
- - - -
- - - - - - -
+ + + +
- - - -
- - - + + + +
- - - - - - -
- - - - - - -
- - - -
- - - -
- - - -
+ + + +
- - - -
- - - - + - -
- - - - + - -
- - - - + - -
- - - - + - -
- - - - + - -
+ + + +
- + - -
- - + -
- - + -
- - + -
- - + -
- - + -
- -
- - - +
- - - +
- - - +
- - - +
- -
- - - +
Fig 7
135
- +
+ -
∗
1200000
1000000
RLU
800000
†
600000
400000
200000
0
ER S167A
+
+
+
+
+
+
+
+
E2
-
+
-
+
-
+
-
+
Dom
-
-
+
+
-
-
+
+
SRC-1
-
-
-
-
+
+
+
+
Fig 8
136
A
c-src
PI3K
ERK1/2
?
P
S118
P
CBP
P
P
SRC-1 P
P
P
S167 PP ER
B
×
S118
AKT
PP
ERPP
c-src
×
ERK1/2
S167
×
PI3K
AKT
P
P
CBP
P
SRC-1
P
ER ER
P
Fig 9
137
Fig S-1
138
56
m
0
+D
om
6
Ta
m
+S
u6
65
Ta
Ta
m
E2
+D
om
†
+D
om
+S
u6
6
∗
Ta
m
m
†
Ta
m
15
m
56
∗
Ta
65
0
†
E2
E2
+S
u6
65
6
C-MYC
E2
+D
o
+S
U
†
E2
†
Do
m
6
n
5
Do
m
B
Su
66
5
Co
10
E2
n
Fold Diffrence
25
Su
66
56
Co
Fold Difference
A
∗
20
15
∗
∗
20
pS2
∗
10
†
∗
5
∗
Input
Fig S-2
139
∗
+S
U6
65
6
4
Ta
m
+S
U6
65
6
Ta
m
Fold Difference
2
Ta
m
6
E2
E2
+S
U6
65
6
SU
66
56
Co
n
Genomic DNA
Con 1°AB
3
Ta
m
C-myc
consesus
ERE
Fold Difference
Input
E2
E2
+S
U6
65
6
Genomic DNA
Con 1°AB
Tam
Tam+SU6656
C-myc
Nonconsesus
ERE
SU
66
56
Co
n
B
Tam
Tam+SU6656
EST
Est+SU6656
SU6656
Con
A
EST
Est+SU6656
SU6656
Con
3.5
†
2.5
†
∗
1.5
∗
1
0.5
0
†
5
†
3
2
∗
1
0
B
6
C-myc
consesus
ERE
Input
Fig S-3
140
3
2
E
E2
+W 2
OR
T
Ta
Ta
m
m
+W
OR
T
W
OR
T
Co
n
Input
Fold Difference
C-myc
Nonconsesus
ERE
Fold Difference
Genomic DNA
Con 1°AB
Tam+WORT
Tam
Est+WORT
EST
WORT
Con
3.5
E
E2
+W 2
OR
T
T
am
Ta
m
+W
OR
T
W
OR
T
Co
n
Genomic DNA
Con 1°AB
Tam+WORT
Tam
Est+WORT
WORT
EST
Con
A
†
3
†
2.5
2
∗
1.5
∗
1
0.5
0
5
†
4
∗
†
∗
1
0
A
C-myc nonConsesus ERE
∗
12
∗
Fold Difference
10
8
6
†
4
†
†
†
†
2
ER wt
B
ER S118A
E2
+D
om
E2
ER S167A
C-myc consensus
ERE
∗
18
Do
m
Co
n
E2
+D
om
E2
Do
m
Co
n
E2
E2
+D
om
Do
m
Co
n
0
16
Fold Difference
14
∗
12
10
8
6
†
4
†
†
†
†
2
Fig S-4
ER wt
ER S118A
141
E2
+D
om
E2
Do
m
Co
n
E2
+D
om
E2
Do
m
Co
n
E2
E2
+D
om
Do
m
Co
n
0
ER S167A
Title: Attenuation of Estrogen Receptor α (ERα) Signaling by Selenium in Breast Cancer
Cells via Downregulation of ERα Gene Expression.
Yatrik M. Shah1, Aparna Kaul1, Yan Dong2, Clement Ip2, and Brian G. Rowan1*.
1
Department of Biochemistry & Cancer Biology Medical College of Ohio, Toledo, OH.
2
Department of Cancer Chemoprevention, Roswell Park Cancer Institute, Buffalo, New
York
Running Title: Selenium Inhibits ER-α Signaling in MCF-7 Cells.
Keywords: Breast cancer, Estrogen Receptor, Selenium, MCF-7
∗
Corresponding author: Brian G. Rowan, Department of Biochemistry & Cancer
Biology, Medical College of Ohio, 3035 Arlington Ave, Toledo, OH 43614-5804; Tel:
419-383-4134; Fax: 419-383-6228; Email: [email protected]
142
Abstract
Numerous studies have shown that selenium provides beneficial effects as a cancer
chemoprevention agent. Although long-term intervention trials failed to confirm
selenium protection against breast cancer in humans because of insufficient cases, the
evidence of effective selenium chemoprevention in animal mammary tumor models or
human breast cancer cells is substantial and convincing. The present study demonstrates
that the selenium compound methylseleninic acid (MSA) inhibits estrogen receptor α
(ERα) signaling in ER-positive MCF-7 breast cancer cells as evidenced by decreased
estradiol-dependent cell growth and gene expression. MSA diminishes estradiol
induction of endogenous ER-regulated pS2 and c-myc genes as well as the expression of
an ER-regulated reporter gene. A major mode of MSA action on ER signaling is through
a downregulation of ERα gene expression that precedes a decrease in ERα protein level.
This study provides a mechanism driven rationale for using selenium as a
chemopreventive agent for women at high risk for developing breast cancer or as a
therapeutic strategy for ER-positive breast cancer.
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Introduction
Breast cancer is the most common female neoplasia in the western world with one
in eight women developing breast cancer in the United States [1]. Estrogens promote the
growth of breast cancer and a strong correlation exists between prolonged estrogen
exposure and breast cancer risk [2]. The major mechanism of estrogen action in breast
tissues is through binding to a specific nuclear estrogen receptor (ER) [3,4]. Two
isoforms of ER have been identified. The role of the major isoform, ERα, in breast
cancer has been clearly defined although the role of the recently discovered ERβ remains
unclear [5-7]. ER is a ligand-activated transcription factor that upon estrogen stimulation
binds to promoter regions of ER-regulated genes to modulate the expression of genes
important for estrogen-regulated cell proliferation [4].
The micronutrient selenium has beneficial effects in inhibiting cancer growth. In
a clinical trial Clark et al. demonstrated the protective effects of selenium against
prostate, lung and colon cancer [8,9]. In addition, selenium inhibits mammary
tumorigenesis [10] and breast-derived cell growth [11]. A recent study demonstrated that
selenium interferes with androgen receptor (AR) signaling to decrease prostate cancer
cell growth [12]. Since the ER belongs to the same superfamily of ligand-activated
nuclear receptors as does the AR, it was of interest to determine whether selenium
compounds could impact ER signaling in breast cancer cells.
144
The present study used methylseleninic acid (MSA), developed specifically for
testing selenium effects in in-vitro cell line experiments [13]. MSA is a monomethylated
selenium compound that bypasses the need for metabolic enzymes required to convert
other selenium species to the active metabolite, methylselenol. This allows MSA, as
opposed to other selenium species to act rapidly in cell lines. Selenomethionine,
currently used in the SELECT clinical trail of prostate cancer, cannot be used in cell
culture studies because most epithelial cell lines lack enzymes needed for metabolism to
the active methylselenol [14]. Our results in the estrogen-dependent MCF-7 breast
cancer cell line demonstrate that MSA inhibits ERα-dependent gene transcription by
decreasing ERα mRNA levels and resulting protein levels. Attenuation of ER signaling
in breast cancer cells is likely a major mechanism contributing to the growth inhibitory
effect of MSA.
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Material and Methods
MTT Assay
MCF-7 cells were plated in 24 well plates (20,000 cells/well) and cultured in
phenol red-free DMEM (Gibco, Grand Island, NY) containing 10% fetal bovine serum
(FBS). Cells were incubated with 1, 5, or 10 µM of MSA at 24 h after plating. An
aliquot of 125 µL of MTT reagent (5 mg/ml of 2,5-diphenyl tetrazolium bromide in PBS)
was pipetted into each well after 24, 48, or 72 h of exposure to MSA. The media with the
MTT reagent was removed after 15-30 min and 300 µL of DMSO (Fisher Biotech,
Fairlawn, NJ) was added to each well. The plates were read at a wavelength of 570 nm.
BrdU Labeling Assay.
Cells were seeded in T75 culture flasks at a density designed to reach 70-80%
confluency at the time of assay. At 48 hr after seeding, cells were exposed to 10 µM
MSA for 16 or 24 hr. During the last 30 min of MSA treatment, cells were labeled with
10 µM of bromodeoxyuridine (10 µl of 1 mM BrdU was added to each ml of culture
media). BrdU-labeled cells were trypsinized, fixed, treated with DNase I, and stained
with fluorescein isothiocyanate (FITC)-conjugated anti-BrdU antibody using the BrdU
Flow Kit from BD Pharmigen (San Diego, CA). Stained cells were then quantified by
flow cytometry, and the data were analyzed with the WinList software (Variety Software
House, Topsham, ME).
146
Detection of Apoptosis.
Cells were seeded in triplicate in a 96-well microtiter plate at a density designed
to reach ~104 cells per well at the time of assay. At 48 hr after seeding, cells were
exposed to either 5 or 10 µM MSA for 24 hr. Detached cells were pulled with attached
cells by centrifugation. Cytoplasmic histone-associated DNA fragments were quantified
using the Cell Death Detection ELISAPLUS Kit (Roche Applied Science, Indianapolis, IN)
as per manufacturer’s protocol. The absorbance measured at 405 nm (with reference
wavelength 492 nm) was normalized by the protein concentration of the samples.
Quantitation of Propidium-Iodide-Stained Cells by Flow Cytometry.
MCF-7 cells were seeded in T75 culture flasks at a density designed to reach 7080% confluency at the time of assay. At 48 hr after seeding, cells were exposed to either
5 or 10 µM MSA for 6, 16 or 24 hr. Adherent cells harvested by mild trypsinization were
pooled together with detached cells (if any). Cells were incubated with 1 µg/ml of
propidium iodide (PI) for 5 min on ice. PI-stained cells were subsequently quantified by
flow cytometry, and the data were analyzed with the WinList software (Variety Software
House, Topsham, ME).
Luciferase Assay
MCF-7 and HeLa cells were plated in 6-well plates (2 ×105 cell/well) and cultured
in phenol red-free DMEM containing 2% FBS that had been charcoal stripped to remove
147
endogenous steroids. At 24 h after plating, the MCF-7 cells were transfected with 500 ng
of EREe1b-luciferase reporter, and the HeLa cells were cotransfected with 50 ng of ERα
and 500 ng EREe1b-luciferase reporter using Fugene transfection reagent (Roche,
Madison, WI). At 24 h post transfection, the cells were incubated with either vehicle,
estradiol (10-8 M) (Sigma, St. Louis, MO), MSA (1, 5 or 10 µM), or a combination of
estradiol and MSA for 24 h. Luciferase expression was measured and normalized as
previously described [15].
Western blot analysis
MCF-7 and HeLa cells were plated in 100 mm dishes (3 ×106 cell/plate), and
cultured in 2% charcoal-stripped FBS in DMEM. MCF-7 cells were maintained in the
stripped media for 3 days until 90% confluency. HeLa cells were transfected at 24h postplating with the expression vector for hERα (0.5 µg /plate) or the empty vector (0.5
µg/plate) and maintained in 2% stripped FBS in DMEM for an additional 48 h. Both
MCF-7 and HeLa cells were incubated with either vehicle, estradiol, MSA or a
combination of estradiol and MSA at varying concentrations and times as indicated in the
figure legends. The cells were lysed and prepared for Western blotting as previously
described [15]. The membranes were incubated with an antibody against ERα
(Novacastra, Newcastle on Tyne, UK), and normalized to β-actin (Santa Cruz
Biotechnology INC, Santa Cruz, CA).
Real-Time RT-PCR
148
The culture and/or transfection conditions for MCF-7 and HeLa cells were
identical to that described above for Western blot analysis. Total mRNA was extracted
from the cell pellet, reverse transcribed and gene expression was measured by real-time
RT-PCR as described previously [15].
C-myc:
FWD-5’-CGTCTCCACACATCAGCACAA -3’
REV-5’-TGTTGGCAGCAGGATAGTCCTT -3’
Probe-5’-56FAM/ACGCAGCGCCTCCCTCCACTC/3BHQ-1/-3’
pS2:
FWD-5’-CGTGAAAGACAGAATTGTGGTTTT-3’
REV-5’- CGTCGAAACAGCAGCCCTTA-3’
Probe-5’-/56FAM/ TGTCACGCCCTCCCAGTGTGCA/3BHQ-1/-3’
ERα
FWD-5’- AGACGGACCAAAGCCACTTG -3’
REV-5’- CCCCGTGATGTAATACTTTTGCA -3’
Probe-5’-/56FAM/ TGCGGGCTCTACTTCATCGCATTCC /3BHQ-1/-3’
ERβ
FWD-5’- CCCAGTGCGCCCTTCAC-3’
149
REV-5’- CAACTCCTTGTCGGCCAACT -3’
Probe-5’-/56FAM/ AGGCCTCCATGATGTCCCTGA /3BHQ-1/-3’
Cofilin
FWD-5’- TGTGCCGGCTGGTTCCT-3’
REV-5’- CTTACTGGTCCTGCTTCCATGAG -3’
Probe-5’-/56FAM/ CTTTTCCCCTGGTCACGGCT /3BHQ-1/-3’
CRABPII
FWD-5’- TTCTCTGGCAACTGGAAAATCA -3’
REV-5’- CATTCACCCCCAGCACTTTG -3’
Probe-5’-/56FAM/ CCGATCGGAAAACTTCGAGGAATTGC /3BHQ-1/-3’
Smooth Muscle Actin
FWD-5’- TCCTCCCTTGAGAAGAGTTACGA -3’
REV-5’- GGCAGCGGAAACGTTCATT -3’
Probe-5’-/56FAM/ TGCCTGATGGGCAAGTGATCA /3BHQ-1/-3’
Data Analysis
Results are expressed as mean ± S.D. P values were calculated using Anova
Dunnett’s T Test and Independent T Test. P<.05 was considered significant.
150
Results
MSA inhibits MCF-7 cell growth via inhibition of proliferation and induction of
apopotosis.
MCF-7 is an ER positive breast cancer cell line that is sensitive to estradiol-induced cell
growth and ER-regulated gene expression. MCF-7 cells were incubated with MSA and
cell growth was measured by the MTT assay. MSA at 5 or 10 µM inhibited cell growth
by approximately 50% at 24, 48, and 72 h (Fig. 1A). To assess the contribution of
apoptosis and anti-proliferation to MSA growth inhibition in MCF-7 cells, BrdU
incorporation and DNA fragmentation were assessed.10µM MSA decreased BrdU
incorporation by 50% and 70% at 16h and 24h, respectively (Fig 1B). In the same time
course, MSA increased apoptosis at 24 h by 8 and 15 fold at 5µM and 10uM MSA,
respectively (Fig. 1C). MSA had no effect on cell toxicity up to 24h as assessed by trypan
blue staining and only very modest effects at 24h as assessed by propidium iodine (PI)
staining (Fig. 1D, top and bottom panel)
MSA inhibits estradiol-dependent induction of estrogen response element (ERE)luciferase reporter gene (ERE2e1b-luciferase) and endogenous ERα-regulated
genes.
To assess whether MSA could alter ER signaling, MCF-7 cells were transfected with the
ERE2e1b-luciferase reporter gene and incubated with estradiol or MSA alone, or coincubated with estradiol and MSA. MSA alone decreased basal luciferase expression and
inhibited estradiol-dependent stimulation of the ERE2e1b-luciferase reporter presence of
151
1, 5, or 10 µM MSA (Fig. 2A). Since MSA inhibited estradiol-dependent induction of
the ERE2e1b-luciferase reporter, it was of interest to determine whether MSA could
inhibit two well-characterized estrogen-regulated genes, c-myc, and pS2. Real-time RT
PCR was used to measure c-myc and pS2 expression after incubation with estradiol alone
or in combination with MSA for 6 h. As expected, estradiol induced pS2 and c-myc
expression and this induction was inhibited in the presence of 1, 2.5, 5, or 10 µM MSA in
a dose-dependent manner (Fig. 2B and C). Additional time course experiments revealed
that estradiol induction of pS2 and c-myc occurred at 2 h and 1 h, respectively (Fig. 2D,
2E). A period of 4 h co-incubation with MSA was required to block the response to
estradiol induction (Fig. 2D and E).
MSA specifically inhibits estradiol-dependent signaling.
In order to rule out the nonspecific effects of MSA on general transcription, MCF-7 cells
were transfected with constitutively active RSV-luciferase reporter and incubated with 1,
5, or 10 µM MSA. No effect on the RSV-luciferase reporter was observed at any MSA
concentration (Fig. 3A). In addition to assess the specificity of MSA towards ER
signaling, MSA effects on several genes that are expressed in MCF-7 cells but unrelated
to estrogen signaling were examined. Cellular retinoic acid binding protein II (CRABPII)
is expressed in MCF-7 cells but is not modulated by estradiol [16]. Cofilin [17] and
smooth muscle actin [18] are expressed in breast cancer cell lines but not regulated by
estradiol (data not shown). MSA had no effect on CRABPII, cofilin, or smooth muscle
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actin mRNA expression (Fig. 3B). These data demonstrate a specificity of MSA towards
estradiol-dependent signaling.
MSA reduces ERα protein expression in MCF-7 cells.
Since MSA inhibited ER signaling, we proceeded to determine if MSA altered ERα
protein levels. MCF-7 cells were incubated with estradiol, MSA, or estradiol + MSA for
6 h, and ERα levels were assessed by Western blot analysis. MSA alone at 5 or 10 µM
reduced ERα level significantly compared to vehicle-incubated samples. Estradiol
treatment alone reduced ERα protein level (Fig 4A, lane 6), most likely due to ubiquitinmediated downregulation as reported previously (15). MSA at 5 or 10 µM further
reduced ERα levels when co-incubated with estradiol (Fig 4A, lanes 9 and 10). To
measure time-dependent decreases of ER by MSA, MCF-7 cells were incubated with 10
µM of MSA for 2, 4, 6, or 12 h. A significant reduction in ERα protein level was
detected after 4 h of incubation with MSA (Fig. 4B); this time frame coincided with the
time it took MSA to inhibit estradiol-dependent induction of pS2 and C-myc (Fig 2D and
E). The effect of MSA on ERβ protein level was also assessed. However, Western
blotting of ERβ in MCF-7 cells generated very weak signals, so the result was
inconclusive (data not shown). To determine whether ERα protein downregulation was a
major mechanism by which MSA inhibits ER signaling, we constitutively expressed ERα
protein in the well-characterized, ER-negative HeLa cell line by transfection with a
153
constitutively expressed ERα expression plasmid. HeLa cells are cervical carcinoma
cells in which reexpression of ERα results in robust estradiol-dependent reporter
activation. Incubation of transfected cells with 10 µM MSA did not affect ERα protein
level (Fig. 4C inset). Under these conditions in which ERα protein levels remained
constant, MSA did not inhibit estradiol induction of the ERE2e1b-luciferase reporter (Fig
3C), nor endogenous pS2 (Fig 4D) or c-myc (Fig. 4E). These results suggest that a major
mechanism contributing to MSA inhibition of estrogen signaling is through reduction in
ERα protein. One caveat to this finding is that HeLa is a cervical carcinoma and not a
breast cancer cell line. Although similar experiments were attempted in two ER-negative
breast cancer cell lines (MDA-MB-231, MDA-MB-468) transfected with ERα, results
were inconclusive since neither cell line displayed estradiol activation of cotransfected
ERE2e1b-luciferase (data not shown).
MSA reduces ERα mRNA.
To determine whether the effect of MSA on ERα was at the transcriptional level, ERα
mRNA in MCF-7 cells was measured by real time RT-PCR following incubation of cells
with estradiol, MSA, or estradiol + MSA. MSA at 5 or 10 µM inhibited ERα gene
expression (Fig. 5A) but had no effect on ERβ gene expression (Fig. 5B). Time course
studies revealed that ERα mRNA was inhibited by 10 µM MSA within 2 h (Fig. 5C),
thereby preceding the decrease in protein level that was not detected until after 4 h, (see
154
Fig. 5B), suggesting that the mechanism by which MSA reduces ERα protein level is
through decreased ERα mRNA transcription.
155
Discussion
ER is a major therapeutic target for hormone-dependent, ER-positive breast
cancers. Antiestrogen therapies designed to compete with estrogen for binding to ER
have proven effective in reducing estrogen-dependent tumor growth. In addition to
targeting ER function, strategies designed to lower ER protein level are also useful in the
management of hormone-dependent, ER-positive tumors. The present study is the first to
describe the disruption of ER signaling in breast cancer cells by a selenium compound.
We found that MSA blocked estradiol-dependent activation of a reporter gene (ERE2e1bluciferase) as well as the transcription of endogenous ER-regulated genes. A major
underlying mechanism by which MSA interferes with ER signaling was through a
decrease in ERα gene transcription and the subsequent reduction of ERα protein.
Several mechanisms may contribute to the growth inhibitory effects of MSA
including antioxidant properties and alteration of redox reactions that may subsequently
induce apoptosis [14,19]. Several independent studies have demonstrated MSA-induced
growth inhibition in in-vitro cell lines via induction of apoptosis. In hyperplastic
mammary epithelium cells TM12 and TM2H [13] and in premalignant human breast cells
MCF10AT1 and MCF10AT3B [20], MSA inhibited cell proliferation and induced
apoptosis. MSA also demonstrated similar effects in human lung cancer cell lines [21]
and human prostate carcinoma cell lines [22,23]. Moreover, MSA effects on cell growth
were not mediated by selenium related toxicity [13] as is detected with inorganic
156
selenium compounds such as sodium selenite and sodium selenide [24]. In addition,
based on our characterization of MSA action in MCF-7 cells, the growth inhibitory
effects of MSA occur through both apoptosis and antiproliferation mechanisms (Fig. 1B
and C). Comprehensive analysis of these mechanisms in a variety or hormone-dependent
and hormone-independent breast cancer cell lines is currently being assessed. What is
demonstrated here for the first time in estrogen-dependent breast cancer cells is a novel
component of the overall antiproliferative effect of MSA; the inhibition of ER signaling.
Estrogens are important mitogenic signals for the growth of breast cancers, and have been
shown to induce G1/S transition in breast cancer cells [25] by control of several key cellcycle regulators ( for ref see [26] ). In addition to inducing cellular proliferation,
estrogens increase cell survival by upregulating the antiapoptotic factor bcl-2 [27] and
downregulating several proapoptotic factors [28]. The overall growth inhibitory effects
of MSA in estrogen dependent breast cancer cells are likely attributed to MSA disruption
of ER signaling and non-ER antiproliferative and apoptotic effects of MSA in hormone
dependent breast cancer. Indeed, MSA also inhibited growth of an ERα-negative breast
cancer cell line (MDA-MB-231, data not shown). Future work is being performed to
assess the relative contribution of loss of ER-signaling to the overall growth inhibitory
effect of MSA in ERα positive breast cancer cells.
Low concentrations of MSA (1 or 2.5 µM) had no significant effect on ERα
protein levels although both concentrations were capable of inhibiting estradioldependent reporter gene activation (Fig. 2A) and endogenous pS2 and c-myc genes (Fig.
157
2B and C). This apparent discrepancy may be attributed to MSA effects on other proteins
important for ERα action. Dong et al. [20] demonstrated that MSA regulates expression
of several proteins shown to be key mediators of ER signaling. For example, MSA
decreased expression of cyclin D1, a known coregulator for ER action [29]. In addition,
MSA decreased the expression of AKT2, a kinase known to phosphorylate ERα resulting
in increased transcriptional activity [30] and stabilization of ERα binding to the pS2 and
c-myc promoters (Shah and Rowan, Molecular Endocrinology, in press).
Although both ERα (present study) and AR [12] gene expression and protein
levels were reduced by MSA, ERβ mRNA expression was unaltered. The lack of
unanimity suggests a specificity of MSA for some, but not all, nuclear receptors. Future
studies will examine the promoter regions of ERα and AR genes to assess whether
homologous transcription factor binding sites are present, and to delineate the promoter
regions required for MSA downregulation of ERα and AR transcription.
Although our study demonstrates a strong MSA inhibition of estrogen signaling
and cell growth in MCF-7 breast cancer cells in vitro, selenium compounds appear to
have negligible effects on proliferation of normal rodent mammary gland [31-33]. This
discrepancy may be due to differences in ERα expression in the normal versus malignant
mammary gland. A very small percentage of epithelial cells in the normal, adult
mammary gland are proliferating and these proliferating cells express very little or no
ERα. In contrast, proliferating, epithelial breast cancer cells are ERα positive [34] and
158
these cells are strong candidates for growth inhibition by antiestrogens [35-37]. If
disruption of ER signaling is one of the major mechanisms for selenium-mediated growth
inhibition, then the normal mammary epithelium may only be modestly affected due to
absence of ERα in the proliferating epithelial cells. These possibilities remain under
intense investigation by our laboratories.
Lastly, the present study presents the possibility that selenium compounds may
not only be useful as a chemopreventative, but may also be efficacious as a therapy for
existing tumors. Several studies have shown growth inhibition of established tumors by
selenium compounds in in-vivo models [33,38-41]. However these studies used inorganic
selenium compounds that are genotoxic and no longer used for selenium
chemoprevention and/or chemotherapy studies. In addition, Yan et al. demonstrated that
supplementation with dietary selenium and magnesium had no effect on HTB123 human
mammary cancer cells inoculated in athymic nude mice [42]. To our knowledge no study
has examined the effects of MSA on established tumors in an in-vivo model. However,
Cao et al. have shown a synergistic interaction of organic selenium compounds with the
anticancer drug irinotecan [43]. Mice bearing squamous cell carcinoma of the head/neck
and colon carcinoma xenografts were given selenium in form of 5-methylselenocysteine
and seleno-L-methionine orally seven days prior to intravenous injection of irinotecan.
Combination treatment of irinotecan + selenium decreased the toxicity of the
chemotherapeutic agent and increased the cure rate of the tumor bearing mice inoculated
with cancer cells sensitive and resistant to irinotecan [43]. Although it is unknown
159
whether selenium monotherapy would be efficacious against MCF-7 xenografts, these
tumors are tamoxifen sensitive [44] suggesting the possibility of combination therapy of
tamoxifen with selenium compounds to improve efficacy.
Tamoxifen is currently the major antiestrogen used for breast cancer therapy, even
though tamoxifen resistance and tamoxifen uterotropic effects represent significant
drawbacks of this modality. ER signaling remains functional in the majority of breast
cancers that exhibit tamoxifen resistance [45]. This suggests that any strategy designed
to disrupt ER signaling or remove ERα protein in tamoxifen-resistant cells may provide
some measure of efficacy. For example the antiestrogen fulvestrant results in ERα
protein degradation and apoptotic cell death. Fulvestrant is recommended for
postmenopausal women who exhibit breast cancer progression following tamoxifen
therapy (for a review see ref.[46]). We are currently assessing whether MSA may be
useful in a similar manner as Fulvestrant since MSA potentiates the growth inhibitory
effects of tamoxifen in tamoxifen sensitive breast cancer cells and resensitize tamoxifen
resistant breast cancer and endometrial cancer cells in vitro (submitted for publication).
An important goal in the endocrine management of hormone dependent breast
cancer is to increase the proportion of cells undergoing apoptotic cell death relative to
cell cycle arrest. Increasing the proportion of cells undergoing apoptosis would prevent
cells from reentering the cell cycle once tamoxifen resistant mechanisms are
acquired resulting in disease recurrence. MSA induces significant apoptosis in MCF-
7 cells. Preclinical therapeutic regimens combining tamoxifen with selenium are being
160
explored for efficacy in preventing or delaying tamoxifen resistance and/or reversing
tamoxifen resistance.
Acknowledgements
Grant Support: This work was supported in part by National Institutes of Health Grant
RO1 DK06832 (to B.G.R.) and Grant R01 CA91990 (to C.I). Y.K.S. was supported by a
Predoctoral Dissertation Award (DISS0100539) from the Susan G. Komen Breast Cancer
Foundation.
161
Reference List
1. Kelsey, J. L. and Berkowitz, G. S.: Breast cancer epidemiology. Cancer Res.
48:5615-5623, 1988
2. Endogenous sex hormones and breast cancer in postmenopausal women: reanalysis
of nine prospective studies. J Natl.Cancer Inst. 94:606-616, 2002
3. Evans, R. M.: The steroid and thyroid hormone receptor superfamily. Science
240:889-895, 1988
4. Tsai, M. J. and O'Malley, B. W.: Molecular mechanisms of action of steroid/thyroid
receptor superfamily members. Annu.Rev.Biochem. 63:451-86.:451-486, 1994
5. Kuiper, G. G., Enmark, E., Pelto-Huikko, M., Nilsson, S., and Gustafsson, J. A.:
Cloning of a novel receptor expressed in rat prostate and ovary.
Proc.Natl.Acad.Sci.U.S.A 93:5925-5930, 1996
6. Mosselman, S., Polman, J., and Dijkema, R.: ER beta: identification and
characterization of a novel human estrogen receptor. FEBS Lett. 19;392:49-53,
1996
7. Tremblay, G. B., Tremblay, A., Copeland, N. G., Gilbert, D. J., Jenkins, N. A.,
Labrie, F., and Giguere, V.: Cloning, chromosomal localization, and functional
analysis of the murine estrogen receptor beta. Mol.Endocrinol. 11:353-365, 1997
8. Duffield-Lillico, A. J., Reid, M. E., Turnbull, B. W., Combs, G. F., Jr., Slate, E. H.,
Fischbach, L. A., Marshall, J. R., and Clark, L. C.: Baseline characteristics and the
effect of selenium supplementation on cancer incidence in a randomized clinical
trial: a summary report of the Nutritional Prevention of Cancer Trial. Cancer
Epidemiol.Biomarkers Prev. 11:630-639, 2002
9. Clark, L. C., Combs, G. F., Jr., Turnbull, B. W., Slate, E. H., Chalker, D. K., Chow,
J., Davis, L. S., Glover, R. A., Graham, G. F., Gross, E. G., Krongrad, A., Lesher, J.
L., Jr., Park, H. K., Sanders, B. B., Jr., Smith, C. L., and Taylor, J. R.: Effects of
selenium supplementation for cancer prevention in patients with carcinoma of the
skin. A randomized controlled trial. Nutritional Prevention of Cancer Study Group.
JAMA 276:1957-1963, 1996
10. el Bayoumy, K., Chae, Y. H., Upadhyaya, P., and Ip, C.: Chemoprevention of
mammary cancer by diallyl selenide, a novel organoselenium compound.
Anticancer Res. 16:2911-2915, 1996
162
11. Redman, C., Scott, J. A., Baines, A. T., Basye, J. L., Clark, L. C., Calley, C., Roe,
D., Payne, C. M., and Nelson, M. A.: Inhibitory effect of selenomethionine on the
growth of three selected human tumor cell lines. Cancer Lett. 125:103-110, 1998
12. Dong, Y., Lee, S. O., Zhang, H., Marshall, J., Gao, A. C., and Ip, C.: Prostate
specific antigen expression is down-regulated by selenium through disruption of
androgen receptor signaling. Cancer Res. 64:19-22, 2004
13. Ip, C., Thompson, H. J., Zhu, Z., and Ganther, H. E.: In vitro and in vivo studies of
methylseleninic acid: evidence that a monomethylated selenium metabolite is
critical for cancer chemoprevention. Cancer Res. 60:2882-2886, 2000
14. Ip, C., Dong, Y., and Ganther, H. E.: New concepts in selenium chemoprevention.
Cancer Metastasis Rev. 21:281-289, 2002
15. Shah, Y. M., Basrur, V., and Rowan, B. G.: Selective estrogen receptor modulator
regulated proteins in endometrial cancer cells. Mol.Cell Endocrinol. 219:127-139,
2004
16. Delva, L., Bastie, J. N., Rochette-Egly, C., Kraiba, R., Balitrand, N., Despouy, G.,
Chambon, P., and Chomienne, C.: Physical and functional interactions between
cellular retinoic acid binding protein II and the retinoic acid-dependent nuclear
complex. Mol.Cell Biol. 19:7158-7167, 1999
17. Bakin, A. V., Safina, A., Rinehart, C., Daroqui, C., Darbary, H., and Helfman, D.
M.: A critical role of tropomyosins in TGF-beta regulation of the actin cytoskeleton
and cell motility in epithelial cells. Mol.Biol.Cell 15:4682-4694, 2004
18. Twal, W. O., Czirok, A., Hegedus, B., Knaak, C., Chintalapudi, M. R., Okagawa,
H., Sugi, Y., and Argraves, W. S.: Fibulin-1 suppression of fibronectin-regulated
cell adhesion and motility. J Cell Sci. 114:4587-4598, 2001
19. Patrick, L.: Selenium biochemistry and cancer: a review of the literature.
Altern.Med.Rev. 9:239-258, 2004
20. Dong, Y., Ganther, H. E., Stewart, C., and Ip, C.: Identification of molecular targets
associated with selenium-induced growth inhibition in human breast cells using
cDNA microarrays. Cancer Res. 62:708-714, 2002
21. Swede, H., Dong, Y., Reid, M., Marshall, J., and Ip, C.: Cell cycle arrest
biomarkers in human lung cancer cells after treatment with selenium in culture.
Cancer Epidemiol.Biomarkers Prev. 12:1248-1252, 2003
163
22. Dong, Y., Zhang, H., Hawthorn, L., Ganther, H. E., and Ip, C.: Delineation of the
molecular basis for selenium-induced growth arrest in human prostate cancer cells
by oligonucleotide array. Cancer Res. 63:52-59, 2003
23. Jiang, C., Wang, Z., Ganther, H., and Lu, J.: Caspases as key executors of methyl
selenium-induced apoptosis (anoikis) of DU-145 prostate cancer cells. Cancer Res.
61:3062-3070, 2001
24. Lu, J., Jiang, C., Kaeck, M., Ganther, H., Vadhanavikit, S., Ip, C., and Thompson,
H.: Dissociation of the genotoxic and growth inhibitory effects of selenium.
Biochem.Pharmacol. 50:213-219, 1995
25. Leung, B. S. and Potter, A. H.: Mode of estrogen action on cell proliferative
kinetics in CAMA-1 cells. I. Effect of serum and estrogen. Cancer Invest 5:187194, 1987
26. Foster, J. S., Henley, D. C., Ahamed, S., and Wimalasena, J.: Estrogens and cellcycle regulation in breast cancer. Trends Endocrinol.Metab 12:320-327, 2001
27. Perillo, B., Sasso, A., Abbondanza, C., and Palumbo, G.: 17beta-estradiol inhibits
apoptosis in MCF-7 cells, inducing bcl-2 expression via two estrogen-responsive
elements present in the coding sequence. Mol.Cell Biol. 20:2890-2901, 2000
28. Frasor, J., Danes, J. M., Komm, B., Chang, K. C., Lyttle, C. R., and
Katzenellenbogen, B. S.: Profiling of estrogen up- and down-regulated gene
expression in human breast cancer cells: insights into gene networks and pathways
underlying estrogenic control of proliferation and cell phenotype. Endocrinology
144:4562-4574, 2003
29. Neuman, E., Ladha, M. H., Lin, N., Upton, T. M., Miller, S. J., DiRenzo, J., Pestell,
R. G., Hinds, P. W., Dowdy, S. F., Brown, M., and Ewen, M. E.: Cyclin D1
stimulation of estrogen receptor transcriptional activity independent of cdk4.
Mol.Cell Biol. 17:5338-5347, 1997
30. Sun, M., Paciga, J. E., Feldman, R. I., Yuan, Z., Coppola, D., Lu, Y. Y., Shelley, S.
A., Nicosia, S. V., and Cheng, J. Q.: Phosphatidylinositol-3-OH Kinase
(PI3K)/AKT2, activated in breast cancer, regulates and is induced by estrogen
receptor alpha (ERalpha) via interaction between ERalpha and PI3K. Cancer Res.
61:5985-5991, 2001
31. Ip, C., Thompson, H. J., and Ganther, H. E.: Selenium modulation of cell
proliferation and cell cycle biomarkers in normal and premalignant cells of the rat
mammary gland. Cancer Epidemiol.Biomarkers Prev. 9:49-54, 2000
164
32. Fico, M. E., Poirier, K. A., Watrach, A. M., Watrach, M. A., and Milner, J. A.:
Differential effects of selenium on normal and neoplastic canine mammary cells.
Cancer Res. 46:3384-3388, 1986
33. Watrach, A. M., Milner, J. A., Watrach, M. A., and Poirier, K. A.: Inhibition of
human breast cancer cells by selenium. Cancer Lett. 25:41-47, 1984
34. Clarke, R. B., Howell, A., Potten, C. S., and Anderson, E.: Dissociation between
steroid receptor expression and cell proliferation in the human breast. Cancer Res.
57:4987-4991, 1997
35. Clarke, R. B., Laidlaw, I. J., Jones, L. J., Howell, A., and Anderson, E.: Effect of
tamoxifen on Ki67 labelling index in human breast tumours and its relationship to
oestrogen and progesterone receptor status. Br.J Cancer 67:606-611, 1993
36. Jordan, V. C. and Koerner, S.: Tamoxifen (ICI 46,474) and the human carcinoma
8S oestrogen receptor. Eur.J.Cancer 11:205-206, 1975
37. Johnston, S. R., MacLennan, K. A., Sacks, N. P., Salter, J., Smith, I. E., and
Dowsett, M.: Modulation of Bcl-2 and Ki-67 expression in oestrogen receptorpositive human breast cancer by tamoxifen. Eur.J Cancer 30A:1663-1669, 1994
38. Watrach, A. M., Milner, J. A., and Watrach, M. A.: Effect of selenium on growth
rate of canine mammary carcinoma cells in athymic nude mice. Cancer Lett.
15:137-143, 1982
39. Poirier, K. A. and Milner, J. A.: Factors influencing the antitumorigenic properties
of selenium in mice. J Nutr. 113:2147-2154, 1983
40. Greeder, G. A. and Milner, J. A.: Factors influencing the inhibitory effect of
selenium on mice inoculated with Ehrlich ascites tumor cells. Science 209:825-827,
1980
41. Medina, D. and Oborn, C. J.: Differential effects of selenium on the growth of
mouse mammary cells in vitro. Cancer Lett. 13:333-344, 1981
42. Yan, L., Boylan, L. M., and Spallholz, J. E.: Effect of dietary selenium and
magnesium on human mammary tumor growth in athymic nude mice. Nutr.Cancer
16:239-248, 1991
43. Cao, S., Durrani, F. A., and Rustum, Y. M.: Selective modulation of the therapeutic
efficacy of anticancer drugs by selenium containing compounds against human
tumor xenografts. Clin.Cancer Res. 10:2561-2569, 2004
165
44. Gottardis, M. M., Robinson, S. P., Satyaswaroop, P. G., and Jordan, V. C.:
Contrasting actions of tamoxifen on endometrial and breast tumor growth in the
athymic mouse. Cancer Res. 48:812-815, 1988
45. Johnston, S. R., Saccani-Jotti, G., Smith, I. E., Salter, J., Newby, J., Coppen, M.,
Ebbs, S. R., and Dowsett, M.: Changes in estrogen receptor, progesterone receptor,
and pS2 expression in tamoxifen-resistant human breast cancer. Cancer Res.
55:3331-3338, 1995
46. McKeage, K., Curran, M. P., and Plosker, G. L.: Fulvestrant: a review of its use in
hormone receptor-positive metastatic breast cancer in postmenopausal women with
disease progression following antiestrogen therapy. Drugs 64:633-648, 2004
166
Figure Legends
Fig. 1. MSA inhibits MCF-7 cell growth through altering proliferation and apoptosis.
(A) Cell growth analysis by MTT assay of MCF-7 cells incubated with vehicle (Veh) or
1, 5, and 10 µM of MSA for 24, 48, and 72 h. Each bar represents the mean value ± S.D.
Vehicle treated samples were set at 100%. (*) = P<.05 compared to vehicle. (B) MCF-7
cell proliferation was measured by BrdU incorporation. MCF-7 cells were incubated with
10µM MSA for 16h and 24 h. BrdU incorporation was measured as described in Material
and Methods, each bar represents the mean value ± S.D. (*) = P<.05 compared to vehicle
(Veh) incubated samples. (C) MCF-7 cells were incubated with 5µM and 10µM MSA for
24h. Following incubation with MSA DNA fragmentation was assessed as described in
Material and Methods, each bar represents the mean value ± S.D. (*) = P<.05 compared
to vehicle (Veh) incubated samples. (D) MCF-7 cells were incubated with 5µM and
10µM MSA for 6, 16, and 24h. Cell toxicity was measured by PI staining (top panel) and
by trypan blue staining (bottom panel) as described in Material and Methods, each bar
represents the mean value ± S.D. (*) = P<.05 compared to vehicle (Veh) incubated
samples.
Fig. 2. MSA inhibits estradiol-dependent activation of an EREe1b-luciferase reporter,
pS2, and c-myc gene expression. (A) MCF-7 cells were transfected with ERE2e1bluciferase reporter (0.5 µg/well). 24 hours posttransfection cells were incubated with
vehicle (Veh), 10-8 M estradiol (E2) or MSA alone or co-incubated with 10-8 M estradiol
and MSA. Standard luciferase assays were performed on cell extracts in triplicate as
167
described in the Materials and Methods. Each bar represents the mean value ± S.D. (*) =
P<.05 compared to estradiol incubated samples alone. Dose response of MSA on (B)
pS2 or (C) c-myc gene expression. MCF-7 cells (2 x 106 cells/plate) were incubated with
vehicle (Veh), 10-8 M estradiol (E2) or MSA alone or co-incubated with 10-8 M estradiol
and MSA for 6 h. Time titration of MSA on (D) pS2 or (E) c-myc gene expression.
MCF-7 cells (2 x 106 cells/plate) were incubated with vehicle (Veh), 10-8 M estradiol
(E2) or 10 µM MSA alone or co-incubated with 10-8 M estradiol (E2) and 10µM MSA.
Expression of (B and D) pS2 and, (C and E) c-myc genes was measured by real time RTPCR as described in the Materials and Methods. Expression was normalized to GAPDH
and each bar represents the mean value ± S.D. (*)= P<.05 compared to vehicle incubated
samples (‡) = P<.05 compared to estradiol (E2) incubated samples.
Fig. 3. MSA specifically inhibits estradiol-dependent signaling. (A) MCF-7 cells were
transfected with RSV-luciferase reporter (0.5 µg/well). 24 hours posttransfection cells
were incubated with vehicle 1, 5, or 10µM MSA. Standard luciferase assays were
performed on cell extracts in triplicate as described in the Materials and Methods. Each
bar represents the mean value ± S.D. (*) = P<.05 compared to estradiol incubated samples
alone. (B) MCF-7 cells (2 x 106 cells/plate) were incubated with vehicle (Veh) or 10 µM
MSA for 2 h. Smooth muscle actin, cofilin and CRABPII genes were measured by real
time RT-PCR as described in the Materials and Methods. Expression was normalized to
GAPDH and each bar represents the mean value ± S.D. (*)= P<.05 compared to vehicle
incubated samples.
168
Fig. 4. MSA inhibits ERα protein expression, which is required for its effect on ERdependent gene expression. (A) Dose response of MSA on ERα protein expression.
MCF-7 cells (2 x 106 cells/plate) were incubated with vehicle (Veh), 10-8 M estradiol
(E2) or MSA alone or co-incubated with 10-8 M estradiol and MSA for 6 h and Western
blot analysis was performed. (B) Time titration of MSA on ERα protein expression.
MCF-7 cells (2 x 106 cells/plate) were incubated with vehicle (Veh) or 10 µM MSA and
Western blot analysis was performed. Quantitation of the Western blot signal for ERα
was normalized to the Western blot signal for β-actin levels (Fig 3A and B, bottom
panels), and each bar represents the mean value ± S.D. (*)= P<.05 compared to vehicle
incubated samples (‡) = P<.05 compared to estradiol (E2) incubated samples. (C) HeLa
cell were transfected with 500 ng of constitutively expressed ER and incubated with
vehicle or 10 µM MSA for 6 h and Western blot analysis was performed for ERα (Inset)
or transfected with 50 ng of constitutively expressed ERα and 500 ng ERE2e1b-luciferase
reporter and incubated with (Veh), 10-8 M estradiol (E2) or 10µM MSA alone or coincubated with 10-8 M estradiol (E2) and 10µM MSA. Standard luciferase assays were
performed on cell extracts in triplicate as described in the Materials and Methods. (*)=
P<.05 compared to vehicle incubated samples (‡) = P<.05 compared to estradiol (E2)
incubated samples. (D and E) HeLa cells were transfected with 500 ng of constitutively
expressed ERα and incubated with (Veh), 10-8 M estradiol (E2) or 10 µM MSA alone or
co-incubated with 10-8 M estradiol (E2) and 10µM MSA for 6 h. Expression of (D) pS2
and, (E) c-myc genes was measured by real time RT-PCR as described in the Materials
169
and Methods. Expression was normalized to GAPDH and each bar represents the mean
value ± S.D. (*)= P<.05 compared to vehicle incubated samples (‡) = P<.05 compared to
estradiol (E2) incubated samples.
Fig. 5. MSA inhibits ERα gene expression. Dose response of MSA on (A) ERα or (B)
ERβ gene expression. MCF-7 cells (2 x 106 cells/plate) were incubated with vehicle
(Veh), 10-8 M estradiol (E2) or MSA alone or co-incubated with 10-8 M estradiol and
MSA for 6 h. (C) Time titration of MSA on ERα gene expression. MCF-7 cells (2 x 106
cells/plate) were incubated with vehicle (Veh) or 10 µM MSA. ERα and ERβ genes
were measured by real time RT-PCR as described in the Materials and Methods.
Expression was normalized to GAPDH and each bar represents the mean value ± S.D.
(*)= P<.05 compared to vehicle incubated samples.
170
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Title: Selenium Disrupts Estrogen Receptor α Signaling and Restores Tamoxifen Sensitivity in
Tamoxifen Resistant Breast and Endometrial Cancer Cells.
Yatrik M. Shah1, Yan Dong2, Clement Ip2, and Brian G. Rowan1,3.
1
Department of Biochemistry & Cancer Biology Medical College of Ohio, Toledo, OH.
Department of Cancer Chemoprevention, Roswell Park Cancer Institute, Buffalo, New
York
2
Running Title: MSA Reverses Tamoxifen Resistance in ERα-positive Cells.
Keywords: Breast cancer, Endometrial cancer, Tamoxifen resistance, Estrogen Receptor,
Selenium
Abbreviation list: Estrogen receptor; ER, Estrogen responsive elements; ERE, Selective
estrogen receptor modulator; SERM, Methylselenocysteine; MSC, Seleno-L-methionine; Se-Met,
Methylseleninic acid; MSA, Dulbecco’s modified Eagle’s medium; DMEM, Fetal bovine serum;
FBS, Fetal calf serum; FCS, Amplified in breast cancer-1; AIB1, Steroid receptor coactivator-1;
SRC-1, Transcriptional intermediary factor-2; TIF2, Nuclear corepressor; N-CoR.
Footnotes:
Grant Support: This work was supported in part by National Institutes of Health Grant
RO1 DK06832 (to B.G.R.) and Grant R01 CA91990 (to C.I). Y.K.S. was supported by a
Predoctoral Dissertation Award (DISS0100539) from the Susan G. Komen Breast Cancer
Foundation.
Acknowledgements: We are grateful to Dr. Robert Clarke for the generous gift of the MCF-7LCC2 cell line.
3
Corresponding author: Brian G. Rowan, Department of Biochemistry & Cancer Biology,
Medical College of Ohio, 3035 Arlington Ave, Toledo, OH 43614-5804; Tel: 419-383-4134; Fax:
419-383-6228; Email: [email protected]
Attenuation of Estrogen Receptor α (ERα) Signaling by Selenium in Breast Cancer Cells via
Downregulation of ERα Gene Expression. Yatrik M. Shah, Aparna Kaul, Yan Dong, Clement Ip,
and Brian G. Rowan. Revised manuscript submitted to Breast Cancer Research and Treatment.
4
180
Abstract
As a treatment for breast cancer, tamoxifen a selective estrogen receptor modulator
(SERM) is the most widely prescribed hormonal therapy for breast cancer. Despite the
benefits of tamoxifen therapy, almost all tamoxifen-responsive breast cancer patients
develop resistance to therapy. In addition, tamoxifen displays estrogen-like effects in the
endometrium increasing the incidence of endometrial cancer. New therapeutic strategies
are needed to circumvent tamoxifen resistance in breast cancer as well as tamoxifen
toxicity in endometrium. Organic selenium compounds are highly effective
chemopreventative agents with well-documented benefits in reducing total cancer
incidence and mortality rates for a number of cancers. The present study demonstrates
that the organic selenium compound methylseleninic acid (MSA, 2.5 µM) can potentiate
growth inhibition of 4-hydroxytamoxifen (10-7 M) in tamoxifen sensitive MCF-7 and
T47D breast cancer cell lines. Remarkably, in tamoxifen resistant MCF-7-LCC2,
HEC1A, and Ishikawa cells, co-incubation of 4-hydroxytamoxifen with MSA resulted in
a marked growth inhibition that was substantially greater than MSA alone. Growth
inhibition by MSA and MSA + 4-hydroxytamoxifen in all cell lines was preceded by a
specific decrease in ERα mRNA and protein without an effect on ERβ levels. Estradiol
and 4-hydroxytamoxifen induction of endogenous ER-dependent gene expression (pS2,
c-myc) as well as ER-dependent reporter gene expression (ERE2e1b-luciferase) was also
attenuated by MSA in all cell lines prior to effect on growth inhibition. Taken together
these data strongly suggest that specific decrease in ERα levels by MSA is required for
both MSA potentiation of the growth inhibitory affects of 4-hydroxytamoxifen and
resensitization of tamoxifen resistant cell lines.
181
Introduction
Estrogens are key hormones for the growth and maintenance of female mammary
gland and reproductive tract and critical for reproduction and fertility. Estrogen binds to
its cognate receptor, the estrogen receptor (ER) belonging to the nuclear receptor
superfamily of ligand dependent transcription factors (1,2). Two different forms of ER
have been characterized, ERα and ERβ, which share high sequence homology (3-5).
Upon ligand binding, the receptor undergoes conformational changes that release ER
from chaperone proteins. Following dimerization, ER binds to estrogen responsive
elements (ERE) in the promoters of ER-dependent genes, and subsequent recruitment of
coactivators initiates ER-dependent gene transcription (6,7).
In addition to maintaining normal reproductive physiology estrogens are
important mitogenic signals in the breast and endometrium, thus implicating the hormone
in breast and endometrial tumorigenesis. As a treatment for breast cancer, tamoxifen a
selective estrogen receptor modulator (SERM), binds to ER and blocks estrogen mediated
breast cancer cell growth (8,9). Tamoxifen is the most widely prescribed endocrine
therapy for breast cancer, and the only agent approved for breast cancer chemoprevention
(10). However, tamoxifen therapy has two major drawbacks. Most tamoxifen responsive
breast cancer patients succumb to tamoxifen resistance (11) in which tumors do not
respond to the growth inhibitory properties of tamoxifen. In addition, tamoxifen displays
estrogen-like effects in the endometrium increasing the incidence of endometrial cancer
(12). Alternative therapeutic strategies that can be used alone or in combination with
182
tamoxifen in ERα positive breast cancers may prove useful in combating tamoxifen
resistance in breast and estrogenic activities in other tissues.
Selenium is an essential micronutrient shown to inhibit cancer growth. Organic
selenium compounds are the agents of choice for chemopreventative studies. These
compounds have fewer side effects and lack the genotoxic action of inorganic selenium
compounds such as selenite (13). Organic selenium agents used in chemoprevention
trials such as methylselenocysteine (MSC) and seleno-L-methionine (Se-Met) are watersoluble compounds that are metabolized in tissues to the active selenium metabolite,
methylselenol (14-16). The clinical usefulness of MSC and Se-Met are in the ability of
these compounds to inhibit DNA synthesis and cell doubling and induce apoptotic cell
death. One of the greatest benefits of organic selenium compounds for chemoprevention
is the very low or absent toxicity (16,17).
In a double-blind placebo-controlled clinical trial Clark et al. demonstrated the
protective effects of selenium-enriched yeast against prostate, lung and colon cancer
(16,18). Although, the study failed to show statistical significance in breast cancer risk
due to insufficient cases there is extensive data demonstrating the growth inhibitory
properties of selenium in breast cancer cell lines and mammary tumor models. Our
previous study using methylseleninic acid (MSA), a rapidly metabolized selenium
compound useful in cell cultures studies (19), demonstrated that MSA inhibits estradiol
induced cell growth and ERα-mediated gene transcription in the ERα-positive MCF-7
breast cancer cell line with no significant toxicity (revised manuscript submitted to Breast
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Cancer Research and Treatment4). The major mechanism by which MSA attenuates ER
signaling was through decrease in ERα mRNA levels and subsequent protein levels with
no effect on ERβ levels. These data suggested a novel mechanism of growth inhibition
by MSA through disruption of estrogen signaling.
Since breast cancers vary widely with regard to ERα expression and de novo
tamoxifen resistance, the present study examined the growth inhibitory mechanisms of
MSA in cell lines that represent different paradigms of ERα expression and tamoxifen
sensitivity/resistance. We show that MSA can inhibit ER signaling and potentiate the
antiestrogen activity of tamoxifen via downregulation of ERα mRNA and protein levels.
MSA in combination with tamoxifen potentiated growth inhibitory properties when
compared to either agent alone in tamoxifen sensitive breast cancer cell lines, and
tamoxifen resistant breast and endometrial cell lines.
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Material and Methods
Cell lines
MCF-7 and MDA-MB-468 were maintained in Dulbecco’s modified Eagle’s
medium (DMEM) (Cellgro, Herndon, VA) supplemented with 10% fetal bovine serum
(FBS) (Gibco, Grand Island, NY), 4 mM of glutamine (Gibco), and 1% penicillinstreptomycin (Gibco) at 37°C with 5% CO2. MCF-7-LCC2 were maintained in IMEM
(Biosource, Rockville, MD) supplemented with 5% fetal calf serum (FCS) (Gibco) that
had been charcoal stripped to remove endogenous steroids and 1%penicillin-streptomycin
at 37°C with 5% CO2. Ishikawa and HEC1A were maintained in DMEM supplemented
with 5% FBS and 1% penicillin-streptomycin at 37°C with 5% CO2.
MTT Assay
MCF-7, MCF-7-LCC2, MDA-MB-468, Ishikawa, and HEC1A cells were plated
in 24 well plates (20,000 cells/well) and cultured in medium described above. Cells were
incubated with MSA (2.5 µM), 4-hydroxytamoxifen (10-7 M) (Sigma, St. Louis, MO) or
combination of 4-hydroxytamoxifen and MSA at 24 h after plating. An aliquot of 125
µL of MTT reagent (ICN, Aurora, OH) (5 mg/ml of 2,5-diphenyl tetrazolium bromide in
PBS) was pipetted into each well after 24, 48, 72, or 96 h post treatment. The media with
the MTT reagent was removed after 30 min- 1h and 300 µL of DMSO (Fisher Biotech,
Fairlawn, NJ) was added to each well. The plates were read at a wavelength of 570 nm.
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Luciferase Assay
MCF-7, Ishikawa, and HEC1A were plated in 6-well plates (2 ×105 cell/well) and
cultured in phenol red-free DMEM containing 2% charcoal-stripped FBS. MCF-7-LCC2
were plated in 6-well plates (2 ×105 cell/well) cultured in phenol red-free IMEM
containing 2% charcoal-stripped FCS. 24 h after plating, MCF-7, MCF-7-LCC2,
Ishikawa, and HEC1A cells were transfected with 500 ng of EREe1b-luciferase reporter
using Fugene transfection reagent (Roche, Madison, WI). 24 h post transfection MCF-7,
Ishikawa, and HEC1A cells were incubated with vehicle, estradiol (10-8 M) (Sigma, St.
Louis, MO), 4-hydroxytamoxifen (10-7 M), MSA (10 µM), or a combination of either
estradiol, tamoxifen, and MSA for 24 h. MCF-7-LCC2 were incubated with vehicle,
estradiol (10-9 M), 4-hydroxytamoxifen (10-8 M), MSA (10 µM), or a combination of
estradiol, tamoxifen and MSA for 24 h. Luciferase expression was measured and
normalized as previously described (20).
Western blot analysis
MCF-7, MCF-7-LCC2, and Ishikawa were plated in 100 mm dishes (3 ×106
cell/plate), and cultured in 2% charcoal-stripped FBS in DMEM. MCF-7-LCC2 were
plated in 100 mm dishes (3 ×106 cell/plate) and cultured in 2% charcoal-stripped FCS.
The cells were maintained in the stripped media for 3 days until 90% confluency. MCF7, MCF-7-LCC2, Ishikawa, and HEC1A were incubated with vehicle, estradiol (10-8 M),
4-hydroxytamoxifen (10-7 M), MSA (10 µM), or a combination of estradiol or 4-
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hydroxytamoxifen and MSA for 6 h. The cells were lysed and prepared for Western
blotting as previously described (20). The membranes were incubated with an antibody
against ERα (Novacastra, Newcastle on Tyne, UK), and normalized to β-actin (Santa
Cruz Biotechnology INC, Santa Cruz, CA).
Real-Time RT-PCR
The culture conditions for MCF-7, MCF-7-LCC2, Ishikawa, and HEC1A were
identical to that described above for Western blot analysis. The cells were incubated with
vehicle, estradiol (10-8 M), 4-hydroxytamoxifen (10-7 M), MSA (10 µM), or a
combination of estradiol or tamoxifen and MSA for 2 h when measuring ERα and ERβ
mRNA expression and 6 h when measuring c-myc and pS2 mRNA expression. Total
mRNA was extracted from the cell pellet, reverse transcribed and gene expression was
measured by real-time RT-PCR as described previously (20).
C-myc:
FWD-5’-CGTCTCCACACATCAGCACAA -3’
REV-5’-TGTTGGCAGCAGGATAGTCCTT -3’
Probe-5’-56FAM/ACGCAGCGCCTCCCTCCACTC/3BHQ-1/-3’
pS2:
FWD-5’-CGTGAAAGACAGAATTGTGGTTTT-3’
REV-5’- CGTCGAAACAGCAGCCCTTA-3’
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Probe-5’-/56FAM/ TGTCACGCCCTCCCAGTGTGCA/3BHQ-1/-3’
ERα
FWD-5’- AGACGGACCAAAGCCACTTG -3’
REV-5’- CCCCGTGATGTAATACTTTTGCA -3’
Probe-5’-/56FAM/ TGCGGGCTCTACTTCATCGCATTCC /3BHQ-1/-3’
ERβ
FWD-5’- CCCAGTGCGCCCTTCAC-3’
REV-5’- CAACTCCTTGTCGGCCAACT -3’
Probe-5’-/56FAM/ AGGCCTCCATGATGTCCCTGA /3BHQ-1/-3’
Ligand Binding Assay
MCF-7 cells were plated in 6-well plates (2 ×105 cell/plate), and cultured in 2%
charcoal stripped FBS in DMEM. The cells were maintained in the stripped media for 3
days until 90% confluent. MCF-7 cells were incubated with MSA (10µM) for 1 h.
Following 1 h incubation 10nM [3H] E2 in presence or absence 500 fold excess cold
estradiol was added to each well and incubated for 1 h. Following 1 h incubation, the
cells were washed 3X with cold 1X PBS. 700µL of ethanol was added to each well and
incubated at room temperature for 30 min. 500uL of ethanolic extract was counted using
liquid scintillation. The relative binding affinity (RBA) was calculated by (Specific
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binding – nonspecific binding/ specific binding) × 100. Vehicle treated samples were set
as 1
Data Analysis
Results are expressed as mean ± S.D. P values were calculated using Anova
Dunnett’s T Test and Independent T Test. P<.05 was considered significant.
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Results
MSA inhibits estradiol- and tamoxifen-dependent activation of an estrogen response
element (ERE)-luciferase reporter gene (ERE2e1b-luciferase).
The effects of MSA on ER signaling were assessed in MCF-7 cells, a well-characterized
ER-positive, tamoxifen sensitive human breast cancer cell, and in MCF-7-LCC2 cells, an
ER-positive tamoxifen resistant variant of the MCF-7 parental line. Two tamoxifen
resistant human endometrial cancer cell lines, Ishikawa and HEC1A, were also assessed
for MSA effects on the ERE2e1b-luciferase. MCF-7, HEC1A, and Ishikawa cells were
transfected with the ERE2e1b-luciferase reporter gene and incubated with estradiol (10-8
M), 4-hydroxytamoxifen (10-7 M), or MSA (10µM) alone, or co-incubated with
combinations of estradiol, 4-hydroxytamoxifen and MSA as indicated (Fig. 1A, C, and
D). MCF-7-LCC2 cells were transfected with the ERE2e1b-luciferase reporter gene and
incubated with estradiol (10-9 M), 4-hydroxytamoxifen (10-8 M), or MSA (10µM) alone,
or co-incubated with combinations of estradiol, 4-hydroxytamoxifen and MSA as
indicated (Fig. 1B). Although MCF-7-LCC2 cells are resistant to tamoxifen growth
inhibition, the cells do not display a true tamoxifen agonist activation of the ERE2e1bluciferase reporter (Fig 1B, Bar 5), an activation that is observed in the endometrial
cancer cell lines HEC1A and Ishikawa (Fig 1C-D, Bar 5) as we and others have
previously reported (20-22). Tamoxifen resistance in MCF-7-LCC2 cells is manifested
by the inability of 4-hydroxytamoxifen to reverse estradiol activation of the reporter (Fig.
1B, Bar 7), which is observed at 10-9 M estradiol + 10-8 M 4-hydroxytamoxifen. In the
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tamoxifen-sensitive MCF-7 parental cells a higher concentration of estradiol (10-8 M)
was required to observe estradiol activation of the reporter. Consequently, a higher
concentration of 4-hydroxytamoxifen (10-7 M) was used for reversing estradiol activation
in MCF-7 cells (Fig. 1A, Bar 7).
To determine whether MSA might impact reporter activation by estradiol and
tamoxifen, cells were co-incubated with estradiol, 4-hydroxtamoxifen, and MSA. MSA
alone decreased basal ERE2e1b-luciferase expression in MCF-7 and MCF-7-LCC2 cells
(Fig. 1A-B, bar 2) and inhibited estradiol-dependent stimulation of the reporter in all cell
lines (Fig. 1A-D, bars 3 and 4). Although MSA did not significantly impact 4hydroxytamoxifen action in either breast cancer cell line (Fig 1A and B, bar 6), MSA
completely blocked 4-hydroxytamoxifen activation of the reporter in both endometrial
cell lines (Fig. 1C and D, bar 6). Co-treatment with MSA potentiated the inhibitory
effect of 4-hydroxytamoxifen on estradiol activation of the reporter in MCF-7 cells (Fig
1A, compare bars 7,8). In MCF-7-LCC2 cells, MSA combined with tamoxifen reversed
tamoxifen resistance by blocking estradiol activation of the reporter greater than the
effect of MSA alone (Fig 1B, compare bars 4,7,8). Note that co-incubation with 4hydroxytamoxifen + estradiol + MSA resulted in greater inhibition of reporter activation
than estradiol + MSA (Fig 1A-B, compare bars 4, 8) or estradiol + 4-hydroxytamoxifen
(Fig 1A-B, compare bars 6, 8). These data demonstrate the MSA inhibits estradiol
activation of the ERE2e1b-luciferase reporter in breast and endometrial cancer cells,
potentiates the antiestrogen effects of 4-hydroxytamoxifen in MCF-7-LCC2 cells, and
blocks 4-hydroxytamoxifen agonist action in endometrial cancer cell lines.
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MSA inhibits the endogenous ERα-regulated gene expression.
To determine whether MSA affected endogenous ER-regulated genes, the wellcharacterized estrogen-regulated gene c-myc was assessed. MCF-7, MCF-7-LCC2,
HEC1A, and Ishikawa were incubated with either vehicle, estradiol (10-8 M), 4hydroxytamoxifen (10-7 M) or MSA (10µM) alone or a combination of estradiol or 4hydroxytamoxifen with MSA for 6 h (Fig. 2A-D). MSA had no effect on basal c-myc
gene expression with the exception of HEC1A cells (fig 2A-D, bar 2). However, MSA
inhibited estradiol-induced gene expression of c-myc in all cell lines (fig 2A-D, bars 3
and 4). 4-hydroxytamoxifen had no effect on basal c-myc gene expression in MCF-7
cells, and MSA in combination with 4-hydroxytamoxifen also had no effect when
compared to vehicle or 4-hydroxytamoxifen incubated samples alone (Fig 2A, bars 5 and
6). In contrast to the inability of 4-hydroxytamoxifen to activate the ERE2e1b-luciferase
reporter in MCF-7-LCC2 cells, 4-hydroxytamoxifen displayed true agonist activation of
endogenous c-myc in these cells (Fig. 2B, bar 5) that was also evident in both
endometrial cell lines HEC1A and Ishikawa (Fig 2C-D, bar 5). MSA blocked 4hydroxytamoxifen activation of c-myc in MCF-7-LCC2, Ishikawa, and HEC1A cells (Fig
2B-D, bar 6). Similar results to those described in Figure 2 were found for the ERdependent pS2 gene (data not shown).
MSA reduces ERα protein levels in tamoxifen-sensitive and –resistant cell lines.
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We previously demonstrated that ERα protein and mRNA downregulation was likely a
major mechanism by which MSA inhibited ER signaling in MCF-7 cells; MSA had no
effect on ERβ mRNA (revised manuscript submitted to Breast Cancer Research and
Treatment4). These experiments were extended to determine whether MSA altered ERα
protein in both tamoxifen-sensitive and resistant cells. In addition, the effect of estradiol
or 4-hydroxytamoxifen alone or in combination with MSA on ERα protein expression
was also assessed. Due to very low expression of ERα in HEC1A cells, the Western blot
analysis was inconclusive (data not shown). MCF-7, MCF-7-LCC2 and Ishikawa cells
were incubated with estradiol (10-8 M), 4-hydroxytamoxifen (10-7 M), or MSA (10µM)
alone, or estradiol or 4-hydroxytamoxifen in combination with MSA for 6 h and ERα
levels were assessed by Western blot analysis. Estradiol treatment alone reduced ERα
protein levels only in the MCF-7 cells (Fig 3A, bar 3) that is likely mediated through the
ubiquitin proteosome pathway (23). In contrast to estradiol, MSA alone significantly
reduced ERα protein in all cell lines (Fig 3A-C, bar 2). MSA further reduced ERα levels
when co-incubated with estradiol in MCF-7 cells (Fig 3A, bars 3 and 4) as we have
previously demonstrated (revised manuscript submitted to Breast Cancer Research and
Treatment4). Remarkably, in MCF-7-LCC2 and Ishikawa cells estradiol had no effect on
ERα protein expression, a previously unreported observation for estradiol regulation of
ERα (Fig. 3B and C, bar 3). Estradiol + MSA reduced ERα protein expression to the
level detected with MSA treatment alone (Fig 3B and C, bars 3 and 4).
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4-hydroxytamoxifen stabilized ERα protein expression in MCF-7 cells (Fig. 3A,
bar 5) as previously reported (24). 4-hydroxytamoxifen also stabilized ERα expression in
Ishikawa cells but had no effect on ERα levels in MCF-7-LCC2 cells (Fig 3B and C,
bar5). Co-incubation of 4-hydroxytamoxifen with MSA decreased ERα expression when
compared to either vehicle or 4-hydroxytamoxifen alone in all cell lines (Fig. 3A-C, bars
5 and 6).
Taken together, these data demonstrate that MSA downregulation of ERα is not
restricted to tamoxifen sensitive MCF-7 cells and occurs in the presence or absence of
ligand. Two unrelated findings from these experiments were that estradiol had no effect
on ERα protein expression in MCF-7-LCC2 and Ishikawa cells, and that 4hydroxytamoxifen stabilized ERα expression in Ishikawa cells (Fig 3C, bar 5), but had
no effect on ERα levels in MCF-7-LCC2 cells (Fig 3B, bar 5). The molecular
mechanisms of cell-specific and ligand-dependent receptor turnover are currently being
investigated.
MSA reduces ERα mRNA but has no affect on ERβ.
Our previous study found that MSA decreased ERα mRNA in MCF-7 cells and the
decrease in mRNA preceded a decrease in ERα protein (revised manuscript submitted to
Breast Cancer Research and Treatment4). We extended these studies to tamoxifen
resistant cell lines and also included assessment of 4-hydroxytamoxifen + MSA. ERα
mRNA in MCF-7, MCF-7-LCC2, HEC1A and Ishikawa cells was measured by real-time
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RT-PCR following incubation of cells with estradiol (10-8 M), 4-hydroxytamoxifen (10-7
M) or MSA (10µM) alone or estradiol or 4-hydroxytamoxifen + MSA for 2 h. As
previously shown, MSA reduced ERα expression in MCF-7 cells (Fig. 4A, bar 2). MSA
also decreased ERα mRNA in MCF-7-LCC2, HEC1A, and Ishikawa cells (Fig. 4B-C,
bar 2). Estradiol or 4-hydroxytamoxifen had no effect on ERα gene expression in MCF7, MCF-7-LCC2, and HEC1A (Fig. 4A-C, bars 3 and 5), although 4-hydroxytamoxifen
significantly decreased ERα mRNA in Ishikawa cells (Fig 4D bar 5). Co-incubation of
estradiol or 4-hydroxytamoxifen with MSA decreased ERα mRNA to levels observed
with MSA alone (Fig. 4A-D, bars 4 and 6). MSA, estradiol, or 4-hydroxytamoxifen
alone or in combination had no effect on ERβ mRNA expression in all cell lines (Fig 5AD) suggesting selective effects of MSA on ERα.
At two hours incubation with MSA, ERα mRNA was reduced with no observed
effects on ERα protein levels (data not shown). This suggests that the MSA-dependent
decrease in ERα mRNA may account for subsequent decrease in ERα protein.
Remarkably, although 4-hydroxtamoxifen decreased ERα mRNA in Ishikawa cells (Fig.
4D, bar 5), ERα protein actually increased with treatment (Fig. 3D, bar 5). The
mechanisms underlying this novel observation are under investigation.
MSA potentiates the growth inhibitory properties of 4-hydroxytamoxifen.
Our previous study found that MSA decreased the growth of MCF-7 cells through a
combination of decreased DNA synthesis and elevated apoptosis (revised manuscript
195
submitted to Breast Cancer Research and Treatment4). MCF-7 cells are also sensitive to
growth inhibition by tamoxifen (25). It was of interest to determine whether coincubation of 4-hydroxytamoxifen with MSA could further potentiate growth inhibition
compared to either agent alone. Furthermore, since long-term tamoxifen treatment is
associated with tamoxifen resistance and endometrial proliferation, it was desirable to
know whether co-incubation of MSA + 4-hydroxytamoxifen could reverse tamoxifen
resistance in breast cancer cells and inhibit tamoxifen-induced endometrial cell
proliferation. MCF-7, MCF-7-LCC2, HEC1A, Ishikawa and an additional tamoxifensensitive ER-positive breast cancer cell line, T47D, were incubated with 4hydroxytamoxifen (10-7 M), MSA (2.5µM) or both agents for 24, 48, 72 and 96 h and
growth was assessed by the MTT assay. Only tamoxifen-sensitive MCF-7 and T47D
cells were growth inhibited by 4-hydroxytamoxifen whereas no effect was observed in
the tamoxifen-resistant MCF-7-LCC2, HEC1A, and Ishikawa cells (Fig. 6A). Increasing
incubation time with 4-hydroxytamoxifen to 9 days resulted in proliferation of HEC1A
and Ishikawa cells (20). In all cell lines, MSA decreased cell growth by 96 hours
incubation (Fig. 6B-F). In tamoxifen sensitive MCF-7 and T47D cells, MSA potentiated
4-hydroxytamoxifen effects on reducing cell growth with the combined treatment more
effective than either agent alone (Fig. 6B and C). Remarkably, in tamoxifen resistant
MCF-7-LCC2, HEC1A and Ishikawa cells in which 4-hydroxytamoxifen alone has no
effect on cell growth, co-incubation of 4-hydroxytamoxifen with MSA resulted in a
marked decrease in cell growth that was substantially greater than MSA treatment alone
(Fig. 6D-F). These results demonstrate that MSA not only potentiates the antiestrogen
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effect of 4-hydroxytamoxifen in tamoxifen-sensitive cells, but MSA also resensitizes
tamoxifen-resistant cells to the growth inhibitory properties of 4-hydroxytamoxifen.
Tamoxifen and MSA do not synergize for growth inhibition in ERα negative cell
lines.
MDA-MB-468 cells, an ERα-negative (26), ERβ-positive (27) human breast cancer cell
line, were incubated with 4-hydroxytamoxifen (10-8 M) for 24, 48, 72, and 96 h and
growth was assessed by the MTT assay. No growth inhibitory effects were observed
after incubation with 4-hydroxytamoxifen (Fig. 7A) as previously reporter (28).
Although MSA (1µM) alone decreased cell growth (Fig. 7A), co-incubation of 4hydroxytamoxifen with MSA did not further decrease cell growth (Fig. 7A) suggesting
that ERα may be required for the additive and/or synergistic effect of tamoxifen + MSA
on cell growth. Similar results were found with the ERα-negative MDA-MB-231 cell
line (data not shown).
Inorganic selenite is reported to interact with the ligand binding domain of ERα
and activate ERα transcriptional activity (29). To determine whether MSA altered
estradiol binding to ERα, whole cell ligand binding assays were performed in MCF-7
cells. One hour incubation of MCF-7 cells with MSA (10µM) did not alter [3H] estradiol
binding to ERα (Fig. 7B).
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Discussion
Tamoxifen resistance and tamoxifen-induced endometrial proliferation are major
limitations of tamoxifen therapy and chemoprevention. The present study demonstrates
that MSA inhibition of ERα signaling is not restricted to tamoxifen sensitive MCF-7
cells. MSA antagonism of estradiol-dependent ERE2e1b-luciferase and endogenous cmyc and pS2 gene expression was demonstrated in MCF-7-LCC2 cells, an ERα-positive,
tamoxifen resistant variant of the MCF-7 parental line and in two ERα-positive human
endometrial cancer cell lines, Ishikawa and HEC1A (Fig. 1 and 2). In addition, MSA
also blocked tamoxifen activation of these genes in endometrial Ishikawa and HEC1A
cells (Fig. 1 and 2 C-D, bar 6). The major mechanism for MSA disruption of ER
signaling in all ERα positive cells lines was via rapid decrease of ERα mRNA and
protein that preceded disruption of ERα-regulated gene expression (Fig. 3 and 4). MSA
alone inhibited the growth of tamoxifen-sensitive and tamoxifen-resistant cells. MSA
also potentiated tamoxifen growth inhibition of ERα-positive, tamoxifen-sensitive cells
(MCF-7 and T47D) and tamoxifen-resistant cells (MCF-7-LCC2, Ishikawa, and HEC1A)
(Fig. 6) but not ERα-negative, tamoxifen resistant cells (MDA-MB-468) (Fig. 7A)
suggesting that ERα is required for the additive and/or synergistic effect on cell growth
inhibition when MSA is combined with tamoxifen.
Interestingly our studies demonstrated that sensitivity to MSA growth inhibition is
cell line specific. HEC1A exhibited a marked decrease in cell growth after treatment
with 2.5 µM MSA for 48 h, whereas MCF-7-LCC2 cells do not show a significant
198
decrease in cell growth until 72 h incubation with MSA (Fig. 6 D and E). In addition,
1µM MSA reduced growth of MDA-MB-468 at 72 h (Fig. 7A), whereas the same
concentration did not affect growth of MCF-7, MCF-7-LCC2, HEC1A, and Ishikawa
cells (data not shown). Currently the cell specific sensitivity of MSA is under
investigation.
A review of the literature from cancer cell lines and in vivo tumors reveals that
MSA and tamoxifen exhibit similarities in growth inhibitory mechanisms. Both agents
induce G1 arrest that is associated with a similar profile of changes in cell cycle
regulatory proteins (19,30-32). Both agents induce a dose dependent apoptosis that is
p53 independent and may involve activation of the same caspases as well as reduction in
bcl-2 (14,15,33-35). This suggests that the added efficacy observed with combination of
selenium with tamoxifen is the result of more pronounced perturbations in several
common regulatory proteins resulting in elevated apoptosis and reduced proliferation
when compared to effects of either agent alone.
As we have previously demonstrated, low concentrations of MSA (1-2.5 µM) had
no effect on ERα mRNA and protein expression while still capable of inhibiting
estradiol-dependent gene expression (revised manuscript submitted to Breast Cancer
Research and Treatment4). Therefore at low MSA concentrations, disruption of ER
signaling occurred via mechanisms independent of ERα depletion suggesting that MSA
also affected ERα function. Dong et al. (35) demonstrated that MSA regulates
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expression of several proteins known to be important mediators of ERα action. In this
study it was shown that MSA decreased cyclin D1 levels in premalignant human breast
cancer cells. In addition to its functions as a cell cycle regulator, cyclin D1 is also an ERα
coactivator (36) and its overexpression is correlated with tamoxifen resistance in ERαpositive postmenopausal breast cancer (37,38). Exogenous expression of cyclin D1 in
tamoxifen sensitive breast cancer cells reverse the growth inhibitory properties of
tamoxifen (39). In addition to cyclin D1, other coactivators are important in the
mechanism of tamoxifen resistance. Overexpression of AIB1 (amplified in breast cancer1 (SRC-3/RAC-3/ACTR)) in patients receiving tamoxifen was correlated with tamoxifen
resistance (40). Elevated SRC-1 (steroid receptor coactivator-1 (NcoA-1)), but not TIF-2
(transcriptional intermediary factor-2 (SRC-2/NcoA-2/GRIP1)) or AIB1 was correlated
with tamoxifen agonist activity in Ishikawa cells (41). Corepressor levels have been
associated with tamoxifen resistance. A decrease in nuclear corepressor (N-CoR) levels
have been associated with a shorter relapse free survival in tamoxifen treated patients,
suggesting that N-CoR may be a good independent prognostic marker of tamoxifen
resistance (42). Dong et al. (35) reported that MSA reduced AKT levels, which is
interesting in light of several reports demonstrating increased AKT activity in tamoxifen
resistant breast caner cells (43,44). Future studies will elucidate the molecular
mechanisms underlying the ability of MSA to restore tamoxifen sensitivity in resistant
cells.
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Although clinical trials with selenium are currently limited to chemoprevention,
recent evidence now strongly demonstrate the potential of utilizing selenium in a new
way; as a novel therapy for overt cancer through combination with well-established
chemotherapeutic and hormonal agents. Several studies have demonstrated growth
inhibition of established tumors by selenium in in vivo models (45-49). However these
studies used inorganic selenium compounds that are genotoxic and no longer used for
selenium chemoprevention. More recently, Cao et al. demonstrated a synergistic
interaction of organic selenium compounds with the topoisomerase 1 poison irinotecan
(50). Xenograft mice bearing squamous cell carcinomas of the head/neck and colon were
given selenium in the form of MSC and Se-Met orally seven days prior to intravenous
injection of irinotecan. Combination treatment of irinotecan + selenium decreased the
toxicity of the chemotherapeutic agent and increased the cure rate of the tumor-bearing
mice inoculated with cancer cells sensitive and resistant to irinotecan (50). The present
study provides a compelling rationale to explore therapeutic regimens combining
tamoxifen with organic selenium compounds for hormone dependent breast cancer.
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Figure Legends
Fig. 1. MSA inhibits estrogen and tamoxifen activated ERE2e1b-luciferase reporter.
(A) MCF-7, (B) MCF-7-LCC2, (C) HEC1A, and (D) Ishikawa cells (2 x 105 cells/well)
were transfected with ERE2e1b-luciferase reporter (0.5 µg/well).
24 hours
posttransfection cells were incubated with vehicle (Veh), 10-8 M estradiol (E2) for MCF7, HEC1A and Ishikawa or 10-9 M E2 for MCF-7-LCC2 cells, 10-7 M 4hydroxytamoxifen (Tam) for MCF-7, HEC1A and Ishikawa or 10-8 M Tam for MCF-7LCC2 cells or 10µM MSA alone or co-incubated with E2 and MSA, Tam and MSA, or
E2, Tam and MSA for 24 h. Standard luciferase assays were performed on cell extracts in
triplicate as described in the Materials and Methods. Each bar represents the mean value
± S.D. (*) = P<.05 compared to vehicle incubated samples. (†) = P<.05 compared to
estradiol or 4-hydroxytamoxifen treated samples. (‡) = P<.05 compared to compared to
estradiol and 4-hydroxtamoxifen co-incubated samples.
Fig. 2. MSA inhibits estradiol and tamoxifen-dependent activation of c-myc gene
expression. (A) MCF-7, (B) MCF-7-LCC2, (C) HEC1A, and (D) Ishikawa cells (2 x 106
cells/plate) were incubated with vehicle (Veh), 10-8 M estradiol (E2) 10-7 M 4hydroxytamoxifen (Tam) or 10µM MSA alone or co-incubated with E2 and MSA or Tam
and MSA for 6 h. C-myc mRNA was measured by real time RT-PCR as described in the
Materials and Methods. Expression was normalized to GAPDH and each bar represents
202
the mean value ± S.D. (*)= P<.05 compared to vehicle incubated samples (†) = P<.05
compared to estradiol or 4-hydroxytamoxifen incubated samples.
Fig. 3. MSA reduces ERα protein. (A) MCF-7, (B) MCF-7-LCC2, and (C) Ishikawa
cells (2 x 106 cells/plate) were incubated with vehicle (Veh), 10-8 M estradiol (E2) 10-7 M
4-hydroxytamoxifen (Tam) or 10µM MSA alone or co-incubated with E2 and MSA or
Tam and MSA for 6 h. Western blot analysis was performed.
Quantitation of the
Western blot signal for ERα was normalized to the Western blot signal for β-actin levels
(Fig 3A,B, and C, Right panels), and each bar represents the mean value ± S.D. (*)=
P<.05 compared to vehicle incubated samples (†) = P<.05 compared to estradiol or 4hydroxtamoxifen incubated samples.
Fig. 4. MSA reduces ERα mRNA. (A) MCF-7, (B) MCF-7-LCC2, (C) HEC1A, and
(D) Ishikawa cells (2 x 106 cells/plate) were incubated with vehicle (Veh), 10-8 M
estradiol (E2), 10-7 M 4-hydroxytamoxifen (Tam), or 10µM MSA alone or co-incubated
with E2 and MSA or Tam and MSA for 2 h. ERα mRNA was measured by real time RTPCR as described in the Materials and Methods. Expression was normalized to GAPDH
and each bar represents the mean value ± S.D. (*)= P<.05 compared to vehicle incubated
samples (†) = P<.05 compared to estradiol or 4-hydroxytamoxifen incubated samples.
Fig. 5. MSA does not alter ERβ mRNA. (A) MCF-7, (B) MCF-7-LCC2, (C) HEC1A,
and (D) Ishikawa cells (2 x 106 cells/plate) were incubated with vehicle (Veh), 10-8 M
203
estradiol (E2), 10-7 M 4-hydroxytamoxifen (Tam), or 10µM MSA alone or co-incubated
with E2 and MSA or Tam and MSA for 2 h. ERβ mRNA was measured by real time RTPCR as described in the Materials and Methods. Expression was normalized to GAPDH
and each bar represents the mean value ± S.D.
Fig. 6. MSA increases the growth inhibitory properties of 4-hydroxytamoxifen. (A)
Growth inhibitory effects of 4-hydroxtamoxifen as measured by MTT assay (A) MCF-7,
T47D, MCF-7-LCC2, HEC1A, and Ishikawa cells (2 x 104 cells/well) were incubated
with vehicle or 10-7 M 4-hydroxytamoxifen for 24, 48, 72, and 96 h. Each bar represents
the mean value ± S.D. The graph is expressed as % vehicle, where vehicle incubated
samples are set as 100% (*) = P<.05 compared to vehicle. (B) MCF-7, (C) T47D, (D)
MCF-7-LCC2, (E) HEC1A, and (F) Ishikawa cells (2 x 104 cells/well) were incubated
with vehicle, 10-7 M 4-hydroxtamoxifen (Tam) or 2.5µM MSA or co-incubated with Tam
and MSA for 24, 48, 72, and 96 h and MTT assays were performed. The graph is
expressed as % vehicle, where vehicle incubated samples are set as 100% (*) = P<.05
compared to 4-hydroxtamoxifen or MSA incubated samples alone.
Fig. 7. Tamoxifen and MSA do not synergize for growth inhibition in ERα negative
cell lines. (A) MDA-MB-468 cells (2 x 104 cells/well) were incubated with vehicle, 10-7
M 4-hydroxtamoxifen (Tam) or 1µM MSA or co-incubated with Tam and MSA for 24,
48, 72, and 96 h and MTT assays were performed. The graph is expressed as % vehicle,
where vehicle incubated samples are set as 100% (B) MSA has no effect on ER ligand
204
binding. MCF-7 cells (4 x 105 cells/well) were incubated with vehicle (Veh) or 10µM
MSA for 1 h. Following 1 h incubation 10nM [3H] E2 in presence or absence 500 fold
excess cold estradiol was added to each well and incubated for 1 h. Relative binding
affinity was measured as described in Material and Methods, and vehicle incubated
samples were set as 1.
205
Reference List
1. Evans, R. M. The steroid and thyroid hormone receptor superfamily. Science 1988.
240: 889-895.
2. Tsai, M. J. and O'Malley, B. W. Molecular mechanisms of action of steroid/thyroid
receptor superfamily members. Annu Rev Biochem 1994. 63:451-86.: 451-486.
3. Kuiper, G. G., Enmark, E., Pelto-Huikko, M., Nilsson, S., and Gustafsson, J. A.
Cloning of a novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci
U S A 1996. 93: 5925-5930.
4. Mosselman, S., Polman, J., and Dijkema, R. ER beta: identification and
characterization of a novel human estrogen receptor. FEBS Lett 1996. 19;392: 4953.
5. Tremblay, G. B., Tremblay, A., Copeland, N. G. et al. Cloning, chromosomal
localization, and functional analysis of the murine estrogen receptor beta. Mol
Endocrinol 1997. 11: 353-365.
6. Klinge, C. M. Estrogen receptor interaction with estrogen response elements.
Nucleic Acids Res 2001. 29: 2905-2919.
7. McKenna, N. J., Lanz, R. B., and O'Malley, B. W. Nuclear receptor coregulators:
cellular and molecular biology. Endocr Rev 1999. 20: 321-344.
8. Dutertre, M. and Smith, C. L. Molecular mechanisms of selective estrogen receptor
modulator (SERM) action. J Pharmacol Exp Ther 2000. 295: 431-437.
9. Jordan, V. C. and Koerner, S. Tamoxifen (ICI 46,474) and the human carcinoma 8S
oestrogen receptor. Eur J Cancer 1975. 11: 205-206.
10. Jordan, V. C. Tamoxifen: a personal retrospective. Lancet Oncol 2000. 1: 43-49.
11. Clarke, R., Leonessa, F., Welch, J. N., and Skaar, T. C. Cellular and molecular
pharmacology of antiestrogen action and resistance. Pharmacol Rev 2001. 53: 2571.
12. Fisher, B., Costantino, J. P., Redmond, C. K. et al. Endometrial cancer in
tamoxifen-treated breast cancer patients: findings from the National Surgical
Adjuvant Breast and Bowel Project (NSABP) B-14. J Natl Cancer Inst 1994. 86:
527-537.
206
13. Sinha, R., Said, T. K., and Medina, D. Organic and inorganic selenium compounds
inhibit mouse mammary cell growth in vitro by different cellular pathways. Cancer
Lett 1996. 107: 277-284.
14. Wang, Z., Jiang, C., and Lu, J. Induction of caspase-mediated apoptosis and cellcycle G1 arrest by selenium metabolite methylselenol. Mol Carcinog 2002. 34: 113120.
15. Ip, C. and Dong, Y. Methylselenocysteine modulates proliferation and apoptosis
biomarkers in premalignant lesions of the rat mammary gland. Anticancer Res
2001. 21: 863-867.
16. Clark, L. C., Combs, G. F., Jr., Turnbull, B. W. et al. Effects of selenium
supplementation for cancer prevention in patients with carcinoma of the skin. A
randomized controlled trial. Nutritional Prevention of Cancer Study Group. JAMA
1996. 276: 1957-1963.
17. Nelson, M. A., Porterfield, B. W., Jacobs, E. T., and Clark, L. C. Selenium and
prostate cancer prevention. Semin Urol Oncol 1999. 17: 91-96.
18. Duffield-Lillico, A. J., Reid, M. E., Turnbull, B. W. et al. Baseline characteristics
and the effect of selenium supplementation on cancer incidence in a randomized
clinical trial: a summary report of the Nutritional Prevention of Cancer Trial.
Cancer Epidemiol Biomarkers Prev 2002. 11: 630-639.
19. Ip, C., Thompson, H. J., Zhu, Z., and Ganther, H. E. In vitro and in vivo studies of
methylseleninic acid: evidence that a monomethylated selenium metabolite is
critical for cancer chemoprevention. Cancer Res 2000. 60: 2882-2886.
20. Shah, Y. M., Basrur, V., and Rowan, B. G. Selective estrogen receptor modulator
regulated proteins in endometrial cancer cells. Mol Cell Endocrinol 2004. 219: 127139.
21. Castro-Rivera, E. and Safe, S. Estrogen- and antiestrogen-responsiveness of
HEC1A endometrial adenocarcinoma cells in culture. J Steroid Biochem Mol Biol
1998. 64: 287-295.
22. Croxtall, J. D., Elder, M. G., and White, J. O. Hormonal control of proliferation in
the Ishikawa endometrial adenocarcinoma cell line. J Steroid Biochem 1990. 35:
665-669.
23. Nawaz, Z., Lonard, D. M., Dennis, A. P., Smith, C. L., and O'Malley, B. W.
Proteasome-dependent degradation of the human estrogen receptor. Proc Natl Acad
Sci U S A 1999. 96: 1858-1862.
207
24. Leclercq, G., Legros, N., and Piccart, M. J. Accumulation of a non-binding form of
estrogen receptor in MCF-7 cells under hydroxytamoxifen treatment. J Steroid
Biochem Mol Biol 1992. 41: 545-552.
25. Jozan, S., Elalamy, H., and Bayard, F. [Mechanism of action of a triphenylethylene
type antiestrogen on growth of the human breast cancer cell line. MCF-7 in culture].
C R Seances Acad Sci III 1981. 292: 767-770.
26. Zugmaier, G., Ennis, B. W., Deschauer, B. et al. Transforming growth factors type
beta 1 and beta 2 are equipotent growth inhibitors of human breast cancer cell lines.
J Cell Physiol 1989. 141: 353-361.
27. Vladusic, E. A., Hornby, A. E., Guerra-Vladusic, F. K., Lakins, J., and Lupu, R.
Expression and regulation of estrogen receptor beta in human breast tumors and cell
lines. Oncol Rep 2000. 7: 157-167.
28. Lindner, D. J. and Borden, E. C. Synergistic antitumor effects of a combination of
interferon and tamoxifen on estrogen receptor-positive and receptor-negative
human tumor cell lines in vivo and in vitro. J Interferon Cytokine Res 1997. 17:
681-693.
29. Stoica, A., Pentecost, E., and Martin, M. B. Effects of selenite on estrogen receptoralpha expression and activity in MCF-7 breast cancer cells. J Cell Biochem 2000.
79: 282-292.
30. Zhu, Z., Jiang, W., Ganther, H. E., and Thompson, H. J. Mechanisms of cell cycle
arrest by methylseleninic acid. Cancer Res 2002. 62: 156-164.
31. Sutherland, R. L., Hall, R. E., and Taylor, I. W. Cell proliferation kinetics of MCF7 human mammary carcinoma cells in culture and effects of tamoxifen on
exponentially growing and plateau-phase cells. Cancer Res 1983. 43: 3998-4006.
32. Sinha, R., Unni, E., Ganther, H. E., and Medina, D. Methylseleninic acid, a potent
growth inhibitor of synchronized mouse mammary epithelial tumor cells in vitro.
Biochem Pharmacol 2001. 61: 311-317.
33. Mandlekar, S., Hebbar, V., Christov, K., and Kong, A. N. Pharmacodynamics of
tamoxifen and its 4-hydroxy and N-desmethyl metabolites: activation of caspases
and induction of apoptosis in rat mammary tumors and in human breast cancer cell
lines. Cancer Res 2000. 60: 6601-6606.
34. Kandouz, M., Siromachkova, M., Jacob, D. et al. Antagonism between estradiol
and progestin on Bcl-2 expression in breast-cancer cells. Int J Cancer 1996. 68:
120-125.
208
35. Dong, Y., Ganther, H. E., Stewart, C., and Ip, C. Identification of molecular targets
associated with selenium-induced growth inhibition in human breast cells using
cDNA microarrays. Cancer Res 2002. 62: 708-714.
36. Neuman, E., Ladha, M. H., Lin, N. et al. Cyclin D1 stimulation of estrogen
receptor transcriptional activity independent of cdk4. Mol Cell Biol 1997. 17: 53385347.
37. Stendahl, M., Kronblad, A., Ryden, L. et al. Cyclin D1 overexpression is a negative
predictive factor for tamoxifen response in postmenopausal breast cancer patients.
Br J Cancer 2004. 90: 1942-1948.
38. Kenny, F. S., Hui, R., Musgrove, E. A. et al. Overexpression of cyclin D1
messenger RNA predicts for poor prognosis in estrogen receptor-positive breast
cancer. Clin Cancer Res 1999. 5: 2069-2076.
39. Wilcken, N. R., Prall, O. W., Musgrove, E. A., and Sutherland, R. L. Inducible
overexpression of cyclin D1 in breast cancer cells reverses the growth-inhibitory
effects of antiestrogens. Clin Cancer Res 1997. 3: 849-854.
40. Osborne, C. K., Bardou, V., Hopp, T. A. et al. Role of the estrogen receptor
coactivator AIB1 (SRC-3) and HER-2/neu in tamoxifen resistance in breast cancer.
J Natl Cancer Inst 2003. 95: 353-361.
41. Shang, Y. and Brown, M. Molecular determinants for the tissue specificity of
SERMs. Science 2002. 295: 2465-2468.
42. Girault, I., Lerebours, F., Amarir, S. et al. Expression analysis of estrogen receptor
alpha coregulators in breast carcinoma: evidence that NCOR1 expression is
predictive of the response to tamoxifen. Clin Cancer Res 2003. 9: 1259-1266.
43. Campbell, R. A., Bhat-Nakshatri, P., Patel, N. M. et al. Phosphatidylinositol 3kinase/AKT-mediated activation of estrogen receptor alpha: a new model for antiestrogen resistance. J Biol Chem 2001. 276: 9817-9824.
44. Clark, A. S., West, K., Streicher, S., and Dennis, P. A. Constitutive and inducible
Akt activity promotes resistance to chemotherapy, trastuzumab, or tamoxifen in
breast cancer cells. Mol Cancer Ther 2002. 1: 707-717.
45. Greeder, G. A. and Milner, J. A. Factors influencing the inhibitory effect of
selenium on mice inoculated with Ehrlich ascites tumor cells. Science 1980. 209:
825-827.
46. Medina, D. and Oborn, C. J. Differential effects of selenium on the growth of
mouse mammary cells in vitro. Cancer Lett 1981. 13: 333-344.
209
47. Poirier, K. A. and Milner, J. A. Factors influencing the antitumorigenic properties
of selenium in mice. J Nutr 1983. 113: 2147-2154.
48. Watrach, A. M., Milner, J. A., Watrach, M. A., and Poirier, K. A. Inhibition of
human breast cancer cells by selenium. Cancer Lett 1984. 25: 41-47.
49. Watrach, A. M., Milner, J. A., and Watrach, M. A. Effect of selenium on growth
rate of canine mammary carcinoma cells in athymic nude mice. Cancer Lett 1982.
15: 137-143.
50. Cao, S., Durrani, F. A., and Rustum, Y. M. Selective modulation of the therapeutic
efficacy of anticancer drugs by selenium containing compounds against human
tumor xenografts. Clin Cancer Res 2004. 10: 2561-2569.
210
*
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4
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Relative Binding Affinty
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Fig 7
222
SUMMARY
The research projects in this dissertation focus on the molecular mechanisms of
tamoxifen sensitivity and resistance with the goal of improving tamoxifen efficacy in
breast and endometrial cancer. These studies have provided protein profiles that may
serve as novel indices of SERM response and may also provide insight into novel
-8
mechanisms of SERM-mediated growth. Following 24-hour incubation with 10
-7
estradiol, 10
M
-7
M 4-hydroxytamoxifen, or 10 M EM-652 (Acolbifene), nine proteins
exhibited significant increase in expression. The proteins identified were heat shock
protein 90 (HSP90)-α, and –β, heterogeneous nuclear ribonucleoprotein F (hnRNP F),
RNA polymerase II-mediating protein (RMP), cytoskeletal keratin 8 (Ck 8), cytoskeletal
keratin 18 (Ck 18), ubiquitin-conjugating enzyme E2-18kDa (UbcH7), and nucleoside
diphosphate kinase B (NDPK B). Interestingly, we have shown NDPK B to be induced
by estradiol and tamoxifen in both the secreted and cytoplasmic fraction of Ishikawa
cells. Previous studies have shown ectopic expression of NDPK B to increase cellular
proliferation (Caligo et al., 1995, 1996). Therefore, future studies will assess whether
NDPK B participates in estrogen- and tamoxifen-dependent proliferation in Ishikawa and
other endometrium-derived cell lines. The NDPK B may serve as a biomarker for the
tamoxifen resistant phenotype, and since NDPK B is a secreted protein it could easily be
assayed by non-invasive procedures.
In addition, these studies present a mechanistic groundwork for rational design of
preclinical therapeutic strategies designed to inhibit src kinase and combine tamoxifen
with organic selenium compounds to circumvent tamoxifen resistant breast cancer and
223
relieve the deleterious estrogen-like side effects of tamoxifen and other SERMs in nontarget tissues. It was found that src kinase inhibitors and MSA disrupted ERα signaling
and potentiated the antagonist action of tamoxifen in sensitive and resistant breast and
endometrial cell lines via separate mechanisms. Src kinase inhibitors blocked ERα
promoter interaction through decrease phosphorylation at serine 167 and inhibited SRC-1
transactivation. Seven phosphorylation sites have been identified on SRC-1 (Rowan et
al., 2000b), although no single site was responsible for src kinase mediated potentiation
of SRC-1. Several possibilities will be assessed in the future: 1) Since all seven SRC-1
phosphorylation sites contain consensus sequences for serine/threonine-proline directed
kinases it is possible that only one or few kinases contribute to steady state SRC-1
phosphorylation; 2) Src kinase increases phosphorylation at multiple sites; 3) Activation
of src kinase mediates a dephosphorylation event on SRC-1; 4) A different protein in the
SRC-1 complex may be a direct or indirect substrate of src kinase; and 5) A novel
phosphorylation site may be involved in src kinase dependent increase in SRC-1 activity.
The MSA decreased ERα mRNA and protein levels in tamoxifen-sensitive and
resistant breast and endometrial cancer cell lines in vitro. Future experiments will
delineate molecular mechanisms modulating MSA decrease of ERα levels by assessing
ERα transcription rates, hnRNA and cytosolic mRNA stability, nuclear mRNA export,
translation rates and protein turnover. A decrease in ERα expression is thought to be the
major mechanism in disruption of ERα-regulated gene expression and potentiation of
tamoxifen-mediated growth inhibition. However, low concentrations of MSA (1-2.5 µM)
had no effect on ERα mRNA and protein expression while still capable of inhibiting
224
estradiol-dependent gene transcription. Therefore at low MSA concentrations, disruption
of ER signaling occurred via mechanisms independent of ERα depletion suggesting that
MSA also affects other parameters of ERα function. The MSA inhibits in vitro DNA
binding by AR (Dong et al., 2004). Dong et al. (2002a) demonstrated that MSA
decreased cyclin D1 levels in premalignant breast neoplasms. In addition to its functions
as a cell cycle regulator, cyclin D1 is also an ERα coactivator (Neuman et al., 1997) and
its overexpression is correlated with tamoxifen resistance in ERα-positive
postmenopausal breast cancer (Kenny et al., 1999; Stendahl et al., 2004). Dong et al.
(Dong et al., 2002a) reported that MSA reduced AKT levels, which have been shown to
be important in tamoxifen resistant breast caner cells (Campbell et al., 2001; Clark et al.,
2002). Future experiments will assess the effects of MSA on ERα promoter interaction
as well as expression levels of other coregulators and kinases.
Future studies will utilize animal models to further validate the SERM-regulated
proteins identified in this dissertation and for the development of non-cross-resistant
therapies utilizing inhibitors of src kinase or selenium in combination with tamoxifen.
The studies presented in the dissertation are particularly important in the prevention and
therapy of breast cancer. By understanding the precise molecular mechanisms better
therapeutic strategies can be developed to inhibit tamoxifen resistant breast cancer and
relieve the deleterious estrogen-like side effects of tamoxifen and other SERMs in nontarget tissues.
225
BIBLIOGRAPHY
(1997). Breast cancer and hormone replacement therapy: collaborative reanalysis of data
from 51 epidemiological studies of 52,705 women with breast cancer and 108,411
women without breast cancer. Collaborative Group on Hormonal Factors in
Breast Cancer. Lancet 350, 1047-1059.
(1998). Tamoxifen for early breast cancer: an overview of the randomised trials. Early
Breast Cancer Trialists' Collaborative Group. Lancet 351, 1451-1467.
(2002). Endogenous sex hormones and breast cancer in postmenopausal women:
reanalysis of nine prospective studies. J Natl Cancer Inst 94, 606-616.
Abram, C. L. and Courtneidge, S. A. (2000). Src family tyrosine kinases and growth
factor signaling. Exp Cell Res 254, 1-13.
Adesanya, O. O., Zhou, J., Samathanam, C., Powell-Braxton, L., and Bondy, C. A.
(1999). Insulin-like growth factor 1 is required for G2 progression in the
estradiol-induced mitotic cycle. Proc Natl Acad Sci U S A 96, 3287-3291.
Agus, D. B., Gordon, M. S., Taylor, C., Natale, R. B., Karlan, B., Mendelson, D. S.,
Press, M. F., Allison, D. E., Sliwkowski, M. X., Lieberman, G., Kelsey, S. M.,
and Fyfe, G. (2005). Phase I Clinical Study of Pertuzumab, a Novel HER
Dimerization Inhibitor, in Patients With Advanced Cancer. J Clin Oncol .
226
Ali, S., Metzger, D., Bornert, J. M., and Chambon, P. (1993). Modulation of
transcriptional activation by ligand-dependent phosphorylation of the human
oestrogen receptor A/B region. EMBO J 12, 1153-1160.
Anderson, L. and Seilhamer, J. (1997). A comparison of selected mRNA and protein
abundances in human liver. Electrophoresis 18, 533-537.
Anzai, Y., Holinka, C. F., Kuramoto, H., and Gurpide, E. (1989). Stimulatory effects of
4-hydroxytamoxifen on proliferation of human endometrial adenocarcinoma cells
(Ishikawa line). Cancer Res 49, 2362-2365.
Anzick, S. L., Kononen, J., Walker, R. L., Azorsa, D. O., Tanner, M. M., Guan, X. Y.,
Sauter, G., Kallioniemi, O. P., Trent, J. M., and Meltzer, P. S. (1997). AIB1, a
steroid receptor coactivator amplified in breast and ovarian cancer. Science 277,
965-968.
Apostolakis, E. M., Ramamurphy, M., Zhou, D., Onate, S., and O'Malley, B. W. (2002).
Acute disruption of select steroid receptor coactivators prevents reproductive
behavior in rats and unmasks genetic adaptation in knockout mice. Mol
Endocrinol 16, 1511-1523.
Arao, Y., Kikuchi, A., Ikeda, K., Nomoto, S., Horiguchi, H., and Kayama, F. (2002).
A+U-rich-element RNA-binding factor 1/heterogeneous nuclear
ribonucleoprotein D gene expression is regulated by oestrogen in the rat uterus.
Biochem J 361, 125-132.
227
Ardizzoni, A. and Tiseo, M. (2004). Second-line chemotherapy in the treatment of
advanced non-small cell lung cancer (NSCLC). J Chemother 16 Suppl 4:104-107.
Arnold, S. F., Obourn, J. D., Jaffe, H., and Notides, A. C. (1994). Serine 167 is the major
estradiol-induced phosphorylation site on the human estrogen receptor. Mol
Endocrinol 8, 1208-1214.
Arnold, S. F., Obourn, J. D., Jaffe, H., and Notides, A. C. (1995a). Phosphorylation of the
human estrogen receptor by mitogen-activated protein kinase and casein kinase II:
consequence on DNA binding. J Steroid Biochem Mol Biol 55, 163-172.
Arnold, S. F., Vorojeikina, D. P., and Notides, A. C. (1995b). Phosphorylation of tyrosine
537 on the human estrogen receptor is required for binding to an estrogen
response element. J Biol Chem 270, 30205-30212.
Arnold, S. F., Obourn, J. D., Jaffe, H., and Notides, A. C. (1995c). Phosphorylation of the
human estrogen receptor on tyrosine 537 in vivo and by src family tyrosine
kinases in vitro. Mol Endocrinol 9, 24-33.
Arnold, S. F., Melamed, M., Vorojeikina, D. P., Notides, A. C., and Sasson, S. (1997).
Estradiol-binding mechanism and binding capacity of the human estrogen
receptor is regulated by tyrosine phosphorylation. Mol Endocrinol 11, 48-53.
Auboeuf, D., Honig, A., Berget, S. M., and O'Malley, B. W. (2002). Coordinate
regulation of transcription and splicing by steroid receptor coregulators. Science
298, 416-419.
228
Baba, H., Urano, T., Okada, K., Furukawa, K., Nakayama, E., Tanaka, H., Iwasaki, K.,
and Shiku, H. (1995). Two isotypes of murine nm23/nucleoside diphosphate
kinase, nm23-M1 and nm23-M2, are involved in metastatic suppression of a
murine melanoma line. Cancer Res 55, 1977-1981.
Bakin, A. V., Safina, A., Rinehart, C., Daroqui, C., Darbary, H., and Helfman, D. M.
(2004). A critical role of tropomyosins in TGF-beta regulation of the actin
cytoskeleton and cell motility in epithelial cells. Mol Biol Cell 15, 4682-4694.
Bakir, S., Mori, T., Durand, J., Chen, Y. F., Thompson, J. A., and Oparil, S. (2000).
Estrogen-induced vasoprotection is estrogen receptor dependent: evidence from
the balloon-injured rat carotid artery model. Circulation 101, 2342-2344.
Balasenthil, S., Barnes, C. J., Rayala, S. K., and Kumar, R. (2004). Estrogen receptor
activation at serine 305 is sufficient to upregulate cyclin D1 in breast cancer cells.
FEBS Lett 567, 243-247.
Benz, C. C., Scott, G. K., Sarup, J. C., Johnson, R. M., Tripathy, D., Coronado, E.,
Shepard, H. M., and Osborne, C. K. (1993). Estrogen-dependent, tamoxifenresistant tumorigenic growth of MCF-7 cells transfected with HER2/neu. Breast
Cancer Res Treat 24, 85-95.
Berry, M., Metzger, D., and Chambon, P. (1990). Role of the two activating domains of
the oestrogen receptor in the cell-type and promoter-context dependent agonistic
activity of the anti-oestrogen 4-hydroxytamoxifen. EMBO J 9, 2811-2818.
229
Berstein, L. M., Zheng, H., Yue, W., Wang, J. P., Lykkesfeldt, A. E., Naftolin, F.,
Harada, H., Shanabrough, M., and Santen, R. J. (2003). New approaches to the
understanding of tamoxifen action and resistance. Endocr Relat Cancer 10, 267277.
Bocchinfuso, W. P. and Korach, K. S. (1997). Mammary gland development and
tumorigenesis in estrogen receptor knockout mice. J Mammary Gland Biol
Neoplasia 2, 323-334.
Bocchinfuso, W. P., Hively, W. P., Couse, J. F., Varmus, H. E., and Korach, K. S.
(1999). A mouse mammary tumor virus-Wnt-1 transgene induces mammary gland
hyperplasia and tumorigenesis in mice lacking estrogen receptor-alpha. Cancer
Res 59, 1869-1876.
Bord, S., Horner, A., Beavan, S., and Compston, J. (2001). Estrogen receptors alpha and
beta are differentially expressed in developing human bone. J Clin Endocrinol
Metab 86, 2309-2314.
Boss, S. M., Huster, W. J., Neild, J. A., Glant, M. D., Eisenhut, C. C., and Draper, M. W.
(1997). Effects of raloxifene hydrochloride on the endometrium of
postmenopausal women. Am J Obstet Gynecol 177, 1458-1464.
Braidman, I. P., Hainey, L., Batra, G., Selby, P. L., Saunders, P. T., and Hoyland, J. A.
(2001). Localization of estrogen receptor beta protein expression in adult human
bone. J Bone Miner Res 16, 214-220.
230
Bression, D., Michard, M., Le Dafniet, M., Pagesy, P., and Peillon, F. (1986). Evidence
for a specific estradiol binding site on rat pituitary membranes. Endocrinology
119, 1048-1051.
Bross, P. F., Baird, A., Chen, G., Jee, J. M., Lostritto, R. T., Morse, D. E., Rosario, L. A.,
Williams, G. M., Yang, P., Rahman, A., Williams, G., and Pazdur, R. (2003).
Fulvestrant in postmenopausal women with advanced breast cancer. Clin Cancer
Res 9, 4309-4317.
Brown, M. T. and Cooper, J. A. (1996). Regulation, substrates and functions of src.
Biochim Biophys Acta 1287, 121-149.
Brubaker, K. D. and Gay, C. V. (1994). Specific binding of estrogen to osteoclast
surfaces. Biochem Biophys Res Commun 200, 899-907.
Buckley, M. M. and Goa, K. L. (1989). Tamoxifen. A reappraisal of its
pharmacodynamic and pharmacokinetic properties, and therapeutic use. Drugs 37,
451-490.
Bunone, G., Briand, P. A., Miksicek, R. J., and Picard, D. (1996). Activation of the
unliganded estrogen receptor by EGF involves the MAP kinase pathway and
direct phosphorylation. EMBO J 15, 2174-2183.
Burakov, D., Wong, C. W., Rachez, C., Cheskis, B. J., and Freedman, L. P. (2000).
Functional interactions between the estrogen receptor and DRIP205, a subunit of
the heteromeric DRIP coactivator complex. J Biol Chem 275, 20928-20934.
231
Burakov, D., Crofts, L. A., Chang, C. P., and Freedman, L. P. (2002). Reciprocal
recruitment of DRIP/mediator and p160 coactivator complexes in vivo by
estrogen receptor. J Biol Chem 277, 14359-14362.
Caligo, M. A., Cipollini, G., Fiore, L., Calvo, S., Basolo, F., Collecchi, P., Ciardiello, F.,
Pepe, S., Petrini, M., and Bevilacqua, G. (1995). NM23 gene expression
correlates with cell growth rate and S-phase. Int J Cancer 60, 837-842.
Caligo, M. A., Cipollini, G., Petrini, M., Valentini, P., and Bevilacqua, G. (1996). Down
regulation of NM23.H1, NM23.H2 and c-myc genes during differentiation
induced by 1,25 dihydroxyvitamin D3. Leuk Res 20, 161-167.
Campbell, R. A., Bhat-Nakshatri, P., Patel, N. M., Constantinidou, D., Ali, S., and
Nakshatri, H. (2001). Phosphatidylinositol 3-kinase/AKT-mediated activation of
estrogen receptor alpha: a new model for anti-estrogen resistance. J Biol Chem
276, 9817-9824.
Cao, S., Durrani, F. A., and Rustum, Y. M. (2004). Selective modulation of the
therapeutic efficacy of anticancer drugs by selenium containing compounds
against human tumor xenografts. Clin Cancer Res 10, 2561-2569.
Cardiff, R. D. and Muller, W. J. (1993). Transgenic mouse models of mammary
tumorigenesis. Cancer Surv 16, 97-113.
Carson, J. H., Kwon, S., and Barbarese, E. (1998). RNA trafficking in myelinating cells.
Curr Opin Neurobiol 8, 607-612.
232
Castano, E., Vorojeikina, D. P., and Notides, A. C. (1997). Phosphorylation of serine-167
on the human oestrogen receptor is important for oestrogen response element
binding and transcriptional activation. Biochem J 326, 149-157.
Castoria, G., Barone, M. V., Di Domenico, M., Bilancio, A., Ametrano, D., Migliaccio,
A., and Auricchio, F. (1999). Non-transcriptional action of oestradiol and
progestin triggers DNA synthesis. EMBO J 18, 2500-2510.
Castoria, G., Migliaccio, A., Bilancio, A., Di Domenico, M., de Falco, A., Lombardi, M.,
Fiorentino, R., Varricchio, L., Barone, M. V., and Auricchio, F. (2001). PI3kinase in concert with Src promotes the S-phase entry of oestradiol-stimulated
MCF-7 cells. EMBO J 20, 6050-6059.
Castro-Rivera, E. and Safe, S. (1998). Estrogen- and antiestrogen-responsiveness of
HEC1A endometrial adenocarcinoma cells in culture. J Steroid Biochem Mol Biol
64, 287-295.
Chakravarti, D., LaMorte, V. J., Nelson, M. C., Nakajima, T., Schulman, I. G., Juguilon,
H., Montminy, M., and Evans, R. M. (1996). Role of CBP/P300 in nuclear
receptor signalling. Nature 383, 99-103.
Chen, D., Ma, H., Hong, H., Koh, S. S., Huang, S. M., Schurter, B. T., Aswad, D. W.,
and Stallcup, M. R. (1999a). Regulation of transcription by a protein
methyltransferase. Science 284, 2174-2177.
233
Chen, D., Pace, P. E., Coombes, R. C., and Ali, S. (1999b). Phosphorylation of human
estrogen receptor alpha by protein kinase A regulates dimerization. Mol Cell Biol
19, 1002-1015.
Chen, H., Lin, R. J., Schiltz, R. L., Chakravarti, D., Nash, A., Nagy, L., Privalsky, M. L.,
Nakatani, Y., and Evans, R. M. (1997). Nuclear receptor coactivator ACTR is a
novel histone acetyltransferase and forms a multimeric activation complex with
P/CAF and CBP/p300. Cell 90, 569-580.
Chen, J., Godt, D., Gunsalus, K., Kiss, I., Goldberg, M., and Laski, F. A. (2001).
Cofilin/ADF is required for cell motility during Drosophila ovary development
and oogenesis. Nat Cell Biol 3, 204-209.
Chen, J. D. and Evans, R. M. (1995). A transcriptional co-repressor that interacts with
nuclear hormone receptors. Nature 377, 454-457.
Choi, I., Gudas, L. J., and Katzenellenbogen, B. S. (2000). Regulation of keratin 19 gene
expression by estrogen in human breast cancer cells and identification of the
estrogen responsive gene region. Mol Cell Endocrinol 164, 225-237.
Chopra, P., Singh, A., Koul, A., Ramachandran, S., Drlica, K., Tyagi, A. K., and Singh,
Y. (2003). Cytotoxic activity of nucleoside diphosphate kinase secreted from
Mycobacterium tuberculosis. Eur J Biochem 270, 625-634.
234
Chu, S., Nishi, Y., Yanase, T., Nawata, H., and Fuller, P. J. (2004). Transrepression of
estrogen receptor beta signaling by nuclear factor-kappab in ovarian granulosa
cells. Mol Endocrinol 18, 1919-1928.
Clark, A. S., West, K., Streicher, S., and Dennis, P. A. (2002). Constitutive and inducible
Akt activity promotes resistance to chemotherapy, trastuzumab, or tamoxifen in
breast cancer cells. Mol Cancer Ther 1, 707-717.
Clark, L. C., Combs, G. F., Jr., Turnbull, B. W., Slate, E. H., Chalker, D. K., Chow, J.,
Davis, L. S., Glover, R. A., Graham, G. F., Gross, E. G., Krongrad, A., Lesher, J.
L., Jr., Park, H. K., Sanders, B. B., Jr., Smith, C. L., and Taylor, J. R. (1996).
Effects of selenium supplementation for cancer prevention in patients with
carcinoma of the skin. A randomized controlled trial. Nutritional Prevention of
Cancer Study Group. JAMA 276, 1957-1963.
Clarke, R., Leonessa, F., Welch, J. N., and Skaar, T. C. (2001). Cellular and molecular
pharmacology of antiestrogen action and resistance. Pharmacol Rev 53, 25-71.
Clarke, R. B., Laidlaw, I. J., Jones, L. J., Howell, A., and Anderson, E. (1993). Effect of
tamoxifen on Ki67 labelling index in human breast tumours and its relationship to
oestrogen and progesterone receptor status. Br J Cancer 67, 606-611.
Clarke, R. B., Howell, A., Potten, C. S., and Anderson, E. (1997). Dissociation between
steroid receptor expression and cell proliferation in the human breast. Cancer Res
57, 4987-4991.
235
Colditz, G. A., Hankinson, S. E., Hunter, D. J., Willett, W. C., Manson, J. E., Stampfer,
M. J., Hennekens, C., Rosner, B., and Speizer, F. E. (1995). The use of estrogens
and progestins and the risk of breast cancer in postmenopausal women. N Engl J
Med 332, 1589-1593.
Coleman, K. M. and Smith, C. L. (2001). Intracellular signaling pathways: nongenomic
actions of estrogens and ligand-independent activation of estrogen receptors.
Front Biosci 6, D1379-91.
Coleman, K. M., Dutertre, M., El Gharbawy, A., Rowan, B. G., Weigel, N. L., and Smith,
C. L. (2003). Mechanistic differences in the activation of estrogen receptor-alpha
(ER alpha)- and ER beta-dependent gene expression by cAMP signaling
pathway(s). J Biol Chem 278, 12834-12845.
Couillard, S., Gutman, M., Labrie, C., Belanger, A., Candas, B., and Labrie, F. (1998).
Comparison of the effects of the antiestrogens EM-800 and tamoxifen on the
growth of human breast ZR-75-1 cancer xenografts in nude mice. Cancer Res 58,
60-64.
Courtneidge, S. A. (2003). Isolation of novel Src substrates. Biochem Soc Trans 31, 2528.
Couse, J. E., Mahato, D., Eddy, E. M., and Korach, K. S. (2001). Molecular mechanism
of estrogen action in the male: insights from the estrogen receptor null mice.
Reprod Fertil Dev 13, 211-219.
236
Couse, J. F., Hewitt, S. C., Bunch, D. O., Sar, M., Walker, V. R., Davis, B. J., and
Korach, K. S. (1999). Postnatal sex reversal of the ovaries in mice lacking
estrogen receptors alpha and beta. Science 286, 2328-2331.
Coutts, A. S., Davie, J. R., Dotzlaw, H., and Murphy, L. C. (1996). Estrogen regulation of
nuclear matrix-intermediate filament proteins in human breast cancer cells. J Cell
Biochem 63, 174-184.
Cowley, S. M., Hoare, S., Mosselman, S., and Parker, M. G. (1997). Estrogen receptors
alpha and beta form heterodimers on DNA. J Biol Chem 272, 19858-19862.
Crombet-Ramos, T., Rak, J., Perez, R., and Viloria-Petit, A. (2002). Antiproliferative,
antiangiogenic and proapoptotic activity of h-R3: A humanized anti-EGFR
antibody. Int J Cancer 20;101, 567-575.
Croxtall, J. D., Elder, M. G., and White, J. O. (1990). Hormonal control of proliferation
in the Ishikawa endometrial adenocarcinoma cell line. J Steroid Biochem 35, 665669.
Cummings, S. R., Eckert, S., Krueger, K. A., Grady, D., Powles, T. J., Cauley, J. A.,
Norton, L., Nickelsen, T., Bjarnason, N. H., Morrow, M., Lippman, M. E., Black,
D., Glusman, J. E., Costa, A., and Jordan, V. C. (1999). The effect of raloxifene
on risk of breast cancer in postmenopausal women: results from the MORE
randomized trial. Multiple Outcomes of Raloxifene Evaluation. JAMA 281, 21892197.
237
Cunha, G. R., Young, P., Hom, Y. K., Cooke, P. S., Taylor, J. A., and Lubahn, D. B.
(1997). Elucidation of a role for stromal steroid hormone receptors in mammary
gland growth and development using tissue recombinants. J Mammary Gland Biol
Neoplasia 2, 393-402.
Davis, B. A. and Cowing, B. E. (2000). Pyridoxal supplementation reduces cell
proliferation and DNA synthesis in estrogen-dependent and -independent
mammary carcinoma cell lines. Nutr Cancer 38, 281-286.
Delaunay, F., Pettersson, K., Tujague, M., and Gustafsson, J. A. (2000). Functional
differences between the amino-terminal domains of estrogen receptors alpha and
beta. Mol Pharmacol 58, 584-590.
Delmas, P. D., Bjarnason, N. H., Mitlak, B. H., Ravoux, A. C., Shah, A. S., Huster, W. J.,
Draper, M., and Christiansen, C. (1997). Effects of raloxifene on bone mineral
density, serum cholesterol concentrations, and uterine endometrium in
postmenopausal women. N Engl J Med 337, 1641-1647.
Delva, L., Bastie, J. N., Rochette-Egly, C., Kraiba, R., Balitrand, N., Despouy, G.,
Chambon, P., and Chomienne, C. (1999). Physical and functional interactions
between cellular retinoic acid binding protein II and the retinoic acid-dependent
nuclear complex. Mol Cell Biol 19, 7158-7167.
238
Desouki, M. M. and Rowan, B. G. (2004). SRC kinase and mitogen-activated protein
kinases in the progression from normal to malignant endometrium. Clin Cancer
Res 10, 546-555.
Dixon, J. M., Anderson, T. J., Page, D. L., Lee, D., and Duffy, S. W. (1982). Infiltrating
lobular carcinoma of the breast. Histopathology 6, 149-161.
Donaldson, S. H., Picher, M., and Boucher, R. C. (2002). Secreted and cell-associated
adenylate kinase and nucleoside diphosphokinase contribute to extracellular
nucleotide metabolism on human airway surfaces. Am J Respir Cell Mol Biol 26,
209-215.
Dong, L., Wang, W., Wang, F., Stoner, M., Reed, J. C., Harigai, M., Samudio, I., Kladde,
M. P., Vyhlidal, C., and Safe, S. (1999). Mechanisms of transcriptional activation
of bcl-2 gene expression by 17beta-estradiol in breast cancer cells. J Biol Chem
274, 32099-32107.
Dong, Y., Ganther, H. E., Stewart, C., and Ip, C. (2002a). Identification of molecular
targets associated with selenium-induced growth inhibition in human breast cells
using cDNA microarrays. Cancer Res 62, 708-714.
Dong, Y., Ip, C., and Ganther, H. (2002b). Evidence of a field effect associated with
mammary cancer chemoprevention by methylseleninic acid. Anticancer Res 22,
27-32.
239
Dong, Y., Zhang, H., Hawthorn, L., Ganther, H. E., and Ip, C. (2003). Delineation of the
molecular basis for selenium-induced growth arrest in human prostate cancer cells
by oligonucleotide array. Cancer Res 63, 52-59.
Dong, Y., Lee, S. O., Zhang, H., Marshall, J., Gao, A. C., and Ip, C. (2004). Prostate
specific antigen expression is down-regulated by selenium through disruption of
androgen receptor signaling. Cancer Res 64, 19-22.
Donovan, J. C., Milic, A., and Slingerland, J. M. (2001). Constitutive MEK/MAPK
activation leads to p27(Kip1) deregulation and antiestrogen resistance in human
breast cancer cells. J Biol Chem 276, 40888-40895.
Dorjsuren, D., Lin, Y., Wei, W., Yamashita, T., Nomura, T., Hayashi, N., and Murakami,
S. (1998). RMP, a novel RNA polymerase II subunit 5-interacting protein,
counteracts transactivation by hepatitis B virus X protein. Mol Cell Biol 18, 75467555.
Drevs, J. (2003). PTK/ZK (Novartis). IDrugs 6, 787-794.
Druker, B. J., Talpaz, M., Resta, D. J., Peng, B., Buchdunger, E., Ford, J. M., Lydon, N.
B., Kantarjian, H., Capdeville, R., Ohno-Jones, S., and Sawyers, C. L. (2001a).
Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in
chronic myeloid leukemia. N Engl J Med 344, 1031-1037.
Druker, B. J., Sawyers, C. L., Kantarjian, H., Resta, D. J., Reese, S. F., Ford, J. M.,
Capdeville, R., and Talpaz, M. (2001b). Activity of a specific inhibitor of the
240
BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and
acute lymphoblastic leukemia with the Philadelphia chromosome. N Engl J Med
344, 1038-1042.
Dubal, D. B., Zhu, H., Yu, J., Rau, S. W., Shughrue, P. J., Merchenthaler, I., Kindy, M.
S., and Wise, P. M. (2001). Estrogen receptor alpha, not beta, is a critical link in
estradiol-mediated protection against brain injury. Proc Natl Acad Sci U S A 98,
1952-1957.
Dubey, R. K., Jackson, E. K., Gillespie, D. G., Zacharia, L. C., Imthurn, B., and Keller,
P. J. (2000). Clinically used estrogens differentially inhibit human aortic smooth
muscle cell growth and mitogen-activated protein kinase activity. Arterioscler
Thromb Vasc Biol 20, 964-972.
Dubik, D. and Shiu, R. P. (1992). Mechanism of estrogen activation of c-myc oncogene
expression. Oncogene 7, 1587-1594.
Duffield-Lillico, A. J., Reid, M. E., Turnbull, B. W., Combs, G. F., Jr., Slate, E. H.,
Fischbach, L. A., Marshall, J. R., and Clark, L. C. (2002). Baseline characteristics
and the effect of selenium supplementation on cancer incidence in a randomized
clinical trial: a summary report of the Nutritional Prevention of Cancer Trial.
Cancer Epidemiol Biomarkers Prev 11, 630-639.
Dutertre, M. and Smith, C. L. (2000). Molecular mechanisms of selective estrogen
receptor modulator (SERM) action. J Pharmacol Exp Ther 295, 431-437.
241
Dutertre, M. and Smith, C. L. (2003). Ligand-independent interactions of p160/steroid
receptor coactivators and CREB-binding protein (CBP) with estrogen receptoralpha: regulation by phosphorylation sites in the A/B region depends on other
receptor domains. Mol Endocrinol 17, 1296-1314.
El Bayoumy, K., Chae, Y. H., Upadhyaya, P., and Ip, C. (1996). Chemoprevention of
mammary cancer by diallyl selenide, a novel organoselenium compound.
Anticancer Res 16, 2911-2915.
El Bayoumy, K. and Sinha, R. (2004). Mechanisms of mammary cancer
chemoprevention by organoselenium compounds. Mutat Res 551, 181-197.
El Tanani, M. K. and Green, C. D. (1997). Two separate mechanisms for ligandindependent activation of the estrogen receptor. Mol Endocrinol 11, 928-937.
Ellis, M. (2004). Overcoming endocrine therapy resistance by signal transduction
inhibition. Oncologist 9 Suppl 3:20-26.
Elsayed, Y. A. and Sausville, E. A. (2001). Selected novel anticancer treatments targeting
cell signaling proteins. Oncologist 6, 517-537.
Endoh, H., Maruyama, K., Masuhiro, Y., Kobayashi, Y., Goto, M., Tai, H., Yanagisawa,
J., Metzger, D., Hashimoto, S., and Kato, S. (1999). Purification and identification
of p68 RNA helicase acting as a transcriptional coactivator specific for the
activation function 1 of human estrogen receptor alpha. Mol Cell Biol 19, 53635372.
242
Enmark, E., Pelto-Huikko, M., Grandien, K., Lagercrantz, S., Lagercrantz, J., Fried, G.,
Nordenskjold, M., and Gustafsson, J. A. (1997). Human estrogen receptor betagene structure, chromosomal localization, and expression pattern. J Clin
Endocrinol Metab 82, 4258-4265.
Esslimani-Sahla, M., Simony-Lafontaine, J., Kramar, A., Lavaill, R., Mollevi, C.,
Warner, M., Gustafsson, J. A., and Rochefort, H. (2004). Estrogen receptor beta
(ER beta) level but not its ER beta cx variant helps to predict tamoxifen resistance
in breast cancer. Clin Cancer Res 10, 5769-5776.
Evans, R. M. (1988). The steroid and thyroid hormone receptor superfamily. Science 240,
889-895.
Fairfield, K. M. and Fletcher, R. H. (2002). Vitamins for chronic disease prevention in
adults: scientific review. JAMA 19;287, 3116-3126.
Feng, W., Webb, P., Nguyen, P., Liu, X., Li, J., Karin, M., and Kushner, P. J. (2001).
Potentiation of estrogen receptor activation function 1 (AF-1) by Src/JNK through
a serine 118-independent pathway. Mol Endocrinol 15, 32-45.
Fico, M. E., Poirier, K. A., Watrach, A. M., Watrach, M. A., and Milner, J. A. (1986).
Differential effects of selenium on normal and neoplastic canine mammary cells.
Cancer Res 46, 3384-3388.
Filardo, E. J., Quinn, J. A., Bland, K. I., and Frackelton, A. R., Jr. (2000). Estrogeninduced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor
243
homolog, GPR30, and occurs via trans-activation of the epidermal growth factor
receptor through release of HB-EGF. Mol Endocrinol 14, 1649-1660.
Filardo, E. J., Quinn, J. A., Frackelton, A. R., Jr., and Bland, K. I. (2002). Estrogen action
via the G protein-coupled receptor, GPR30: stimulation of adenylyl cyclase and
cAMP-mediated attenuation of the epidermal growth factor receptor-to-MAPK
signaling axis. Mol Endocrinol 16, 70-84.
Fisher, B., Costantino, J. P., Redmond, C. K., Fisher, E. R., Wickerham, D. L., and
Cronin, W. M. (1994). Endometrial cancer in tamoxifen-treated breast cancer
patients: findings from the National Surgical Adjuvant Breast and Bowel Project
(NSABP) B-14. J Natl Cancer Inst 86, 527-537.
Fondell, J. D., Ge, H., and Roeder, R. G. (1996). Ligand induction of a transcriptionally
active thyroid hormone receptor coactivator complex. Proc Natl Acad Sci U S A
93, 8329-8333.
Font, de Mora and Brown, M. (2000). AIB1 is a conduit for kinase-mediated growth
factor signaling to the estrogen receptor. Mol Cell Biol 20, 5041-5047.
Fornander, T., Rutqvist, L. E., Cedermark, B., Glas, U., Mattsson, A., Silfversward, C.,
Skoog, L., Somell, A., Theve, T., Wilking, N., and . (1989). Adjuvant tamoxifen
in early breast cancer: occurrence of new primary cancers. Lancet 1, 117-120.
Foster, J. S., Henley, D. C., Ahamed, S., and Wimalasena, J. (2001). Estrogens and cellcycle regulation in breast cancer. Trends Endocrinol Metab 12, 320-327.
244
Frasor, J., Danes, J. M., Komm, B., Chang, K. C., Lyttle, C. R., and Katzenellenbogen, B.
S. (2003). Profiling of estrogen up- and down-regulated gene expression in human
breast cancer cells: insights into gene networks and pathways underlying
estrogenic control of proliferation and cell phenotype. Endocrinology 144, 45624574.
Fujimoto, N. and Katzenellenbogen, B. S. (1994). Alteration in the agonist/antagonist
balance of antiestrogens by activation of protein kinase A signaling pathways in
breast cancer cells: antiestrogen selectivity and promoter dependence. Mol
Endocrinol 8, 296-304.
Gee, J. M., Robertson, J. F., Ellis, I. O., and Nicholson, R. I. (2001). Phosphorylation of
ERK1/2 mitogen-activated protein kinase is associated with poor response to antihormonal therapy and decreased patient survival in clinical breast cancer. Int J
Cancer 20;95, 247-254.
Gehin, M., Mark, M., Dennefeld, C., Dierich, A., Gronemeyer, H., and Chambon, P.
(2002). The function of TIF2/GRIP1 in mouse reproduction is distinct from those
of SRC-1 and p/CIP. Mol Cell Biol 22, 5923-5937.
Girault, I., Lerebours, F., Amarir, S., Tozlu, S., Tubiana-Hulin, M., Lidereau, R., and
Bieche, I. (2003). Expression analysis of estrogen receptor alpha coregulators in
breast carcinoma: evidence that NCOR1 expression is predictive of the response
to tamoxifen. Clin Cancer Res 9, 1259-1266.
245
Gottardis, M. M., Robinson, S. P., Satyaswaroop, P. G., and Jordan, V. C. (1988).
Contrasting actions of tamoxifen on endometrial and breast tumor growth in the
athymic mouse. Cancer Res 48, 812-815.
Grabowski, P. J. (1998). Splicing regulation in neurons: tinkering with cell-specific
control. Cell 20;92, 709-712.
Gradishar, W., Glusman, J., Lu, Y., Vogel, C., Cohen, F. J., and Sledge, G. W., Jr.
(2000). Effects of high dose raloxifene in selected patients with advanced breast
carcinoma. Cancer 88, 2047-2053.
Graham, J., Muhsin, M., and Kirkpatrick, P. (2004). Cetuximab. Nat Rev Drug Discov 3,
549-550.
Greeder, G. A. and Milner, J. A. (1980). Factors influencing the inhibitory effect of
selenium on mice inoculated with Ehrlich ascites tumor cells. Science 209, 825827.
Gu, W., Malik, S., Ito, M., Yuan, C. X., Fondell, J. D., Zhang, X., Martinez, E., Qin, J.,
and Roeder, R. G. (1999). A novel human SRB/MED-containing cofactor
complex, SMCC, involved in transcription regulation. Mol Cell 3, 97-108.
Hall, J. M. and McDonnell, D. P. (1999). The estrogen receptor beta-isoform (ERbeta) of
the human estrogen receptor modulates ERalpha transcriptional activity and is a
key regulator of the cellular response to estrogens and antiestrogens.
Endocrinology 140, 5566-5578.
246
Harper, M. J. and Walpole, A. L. (1967). A new derivative of triphenylethylene: effect on
implantation and mode of action in rats. J Reprod Fertil 13, 101-119.
Heery, D. M., Kalkhoven, E., Hoare, S., and Parker, M. G. (1997). A signature motif in
transcriptional co-activators mediates binding to nuclear receptors. Nature 387,
733-736.
Heinzel, T., Lavinsky, R. M., Mullen, T. M., Soderstrom, M., Laherty, C. D., Torchia, J.,
Yang, W. M., Brard, G., Ngo, S. D., Davie, J. R., Seto, E., Eisenman, R. N., Rose,
D. W., Glass, C. K., and Rosenfeld, M. G. (1997). A complex containing N-CoR,
mSin3 and histone deacetylase mediates transcriptional repression. Nature 387,
43-48.
Hewitt, S. C., Bocchinfuso, W. P., Zhai, J., Harrell, C., Koonce, L., Clark, J., Myers, P.,
and Korach, K. S. (2002). Lack of ductal development in the absence of
functional estrogen receptor alpha delays mammary tumor formation induced by
transgenic expression of ErbB2/neu. Cancer Res 62, 2798-2805.
Hiroi, H., Inoue, S., Watanabe, T., Goto, W., Orimo, A., Momoeda, M., Tsutsumi, O.,
Taketani, Y., and Muramatsu, M. (1999). Differential immunolocalization of
estrogen receptor alpha and beta in rat ovary and uterus. J Mol Endocrinol 22, 3744.
Homma, H., Kurachi, H., Nishio, Y., Takeda, T., Yamamoto, T., Adachi, K., Morishige,
K., Ohmichi, M., Matsuzawa, Y., and Murata, Y. (2000). Estrogen suppresses
247
transcription of lipoprotein lipase gene. Existence of a unique estrogen response
element on the lipoprotein lipase promoter. J Biol Chem 275, 11404-11411.
Hong, H., Kohli, K., Trivedi, A., Johnson, D. L., and Stallcup, M. R. (1996). GRIP1, a
novel mouse protein that serves as a transcriptional coactivator in yeast for the
hormone binding domains of steroid receptors. Proc Natl Acad Sci U S A 93,
4948-4952.
Hong, S. H., Wong, C. W., and Privalsky, M. L. (1998). Signaling by tyrosine kinases
negatively regulates the interaction between transcription factors and SMRT
(silencing mediator of retinoic acid and thyroid hormone receptor) corepressor.
Mol Endocrinol 12, 1161-1171.
Hong, S. H. and Privalsky, M. L. (2000). The SMRT corepressor is regulated by a MEK1 kinase pathway: inhibition of corepressor function is associated with SMRT
phosphorylation and nuclear export. Mol Cell Biol 20, 6612-6625.
Hopp, T. A., Weiss, H. L., Parra, I. S., Cui, Y., Osborne, C. K., and Fuqua, S. A. (2004).
Low levels of estrogen receptor beta protein predict resistance to tamoxifen
therapy in breast cancer. Clin Cancer Res 10, 7490-7499.
Horlein, A. J., Naar, A. M., Heinzel, T., Torchia, J., Gloss, B., Kurokawa, R., Ryan, A.,
Kamei, Y., Soderstrom, M., Glass, C. K., and . (1995). Ligand-independent
repression by the thyroid hormone receptor mediated by a nuclear receptor corepressor. Nature 377, 397-404.
248
Howell, A., DeFriend, D., Robertson, J., Blamey, R., and Walton, P. (1995). Response to
a specific antioestrogen (ICI 182780) in tamoxifen-resistant breast cancer. Lancet
345, 29-30.
Howell, A., Cuzick, J., Baum, M., Buzdar, A., Dowsett, M., Forbes, J. F., Hoctin-Boes,
G., Houghton, J., Locker, G. Y., and Tobias, J. S. (2005). Results of the ATAC
(Arimidex, Tamoxifen, Alone or in Combination) trial after completion of 5 years'
adjuvant treatment for breast cancer. Lancet 365, 60-62.
Hu, X. and Lazar, M. A. (1999). The CoRNR motif controls the recruitment of
corepressors by nuclear hormone receptors. Nature 402, 93-96.
Hulka, B. S., Liu, E. T., and Lininger, R. A. (1994). Steroid hormones and risk of breast
cancer. Cancer 74, 1111-1124.
Ip, C. and Lisk, D. J. (1994). Enrichment of selenium in allium vegetables for cancer
prevention. Carcinogenesis 15, 1881-1885.
Ip, C., Zhu, Z., Thompson, H. J., Lisk, D., and Ganther, H. E. (1999). Chemoprevention
of mammary cancer with Se-allylselenocysteine and other selenoamino acids in
the rat. Anticancer Res 19, 2875-2880.
Ip, C., Thompson, H. J., Zhu, Z., and Ganther, H. E. (2000a). In vitro and in vivo studies
of methylseleninic acid: evidence that a monomethylated selenium metabolite is
critical for cancer chemoprevention. Cancer Res 60, 2882-2886.
249
Ip, C., Thompson, H. J., and Ganther, H. E. (2000b). Selenium modulation of cell
proliferation and cell cycle biomarkers in normal and premalignant cells of the rat
mammary gland. Cancer Epidemiol Biomarkers Prev 9, 49-54.
Ip, C. and Dong, Y. (2001). Methylselenocysteine modulates proliferation and apoptosis
biomarkers in premalignant lesions of the rat mammary gland. Anticancer Res 21,
863-867.
Ip, C., Dong, Y., and Ganther, H. E. (2002). New concepts in selenium chemoprevention.
Cancer Metastasis Rev 21, 281-289.
Irby, R. B. and Yeatman, T. J. (2000). Role of Src expression and activation in human
cancer. Oncogene 20;19, 5636-5642.
Jakacka, M., Ito, M., Martinson, F., Ishikawa, T., Lee, E. J., and Jameson, J. L. (2002).
An estrogen receptor (ER)alpha deoxyribonucleic acid-binding domain knock-in
mutation provides evidence for nonclassical ER pathway signaling in vivo. Mol
Endocrinol 16, 2188-2201.
Jepsen, K., Hermanson, O., Onami, T. M., Gleiberman, A. S., Lunyak, V., McEvilly, R.
J., Kurokawa, R., Kumar, V., Liu, F., Seto, E., Hedrick, S. M., Mandel, G., Glass,
C. K., Rose, D. W., and Rosenfeld, M. G. (2000). Combinatorial roles of the
nuclear receptor corepressor in transcription and development. Cell 102, 753-763.
Jepsen, K. and Rosenfeld, M. G. (2002). Biological roles and mechanistic actions of corepressor complexes. J Cell Sci 115, 689-698.
250
Jessop, H. L., Suswillo, R. F., Rawlinson, S. C., Zaman, G., Lee, K., Das-Gupta, V.,
Pitsillides, A. A., and Lanyon, L. E. (2004). Osteoblast-like cells from estrogen
receptor alpha knockout mice have deficient responses to mechanical strain. J
Bone Miner Res 19, 938-946.
Jiang, C., Wang, Z., Ganther, H., and Lu, J. (2001). Caspases as key executors of methyl
selenium-induced apoptosis (anoikis) of DU-145 prostate cancer cells. Cancer Res
61, 3062-3070.
Jiang, C., Wang, Z., Ganther, H., and Lu, J. (2002). Distinct effects of methylseleninic
acid versus selenite on apoptosis, cell cycle, and protein kinase pathways in
DU145 human prostate cancer cells. Mol Cancer Ther 1, 1059-1066.
Jiang, S. Y., Langan-Fahey, S. M., Stella, A. L., McCague, R., and Jordan, V. C. (1992).
Point mutation of estrogen receptor (ER) in the ligand-binding domain changes
the pharmacology of antiestrogens in ER-negative breast cancer cells stably
expressing complementary DNAs for ER. Mol Endocrinol 6, 2167-2174.
Jing, Y., Waxman, S., and Lopez, R. (1997). The cellular retinoic acid binding protein II
is a positive regulator of retinoic acid signaling in breast cancer cells. Cancer Res
57, 1668-1672.
Johnston, S. R., MacLennan, K. A., Sacks, N. P., Salter, J., Smith, I. E., and Dowsett, M.
(1994). Modulation of Bcl-2 and Ki-67 expression in oestrogen receptor-positive
human breast cancer by tamoxifen. Eur J Cancer 30A, 1663-1669.
251
Johnston, S. R., Saccani-Jotti, G., Smith, I. E., Salter, J., Newby, J., Coppen, M., Ebbs, S.
R., and Dowsett, M. (1995). Changes in estrogen receptor, progesterone receptor,
and pS2 expression in tamoxifen-resistant human breast cancer. Cancer Res 55,
3331-3338.
Jordan, V. C. and Koerner, S. (1975). Tamoxifen (ICI 46,474) and the human carcinoma
8S oestrogen receptor. Eur J Cancer 11, 205-206.
Jordan, V. C. (1982). Metabolites of tamoxifen in animals and man: identification,
pharmacology, and significance. Breast Cancer Res Treat 2, 123-138.
Jordan, V. C. (2000). Tamoxifen: a personal retrospective. Lancet Oncol 1, 43-49.
Jozan, S., Elalamy, H., and Bayard, F. (1981). Mechanism of action of a
triphenylethylene type antiestrogen on growth of the human breast cancer cell
line. MCF-7 in culture. C R Seances Acad Sci III 292, 767-770.
Kahlert, S., Nuedling, S., van Eickels, M., Vetter, H., Meyer, R., and Grohe, C. (2000).
Estrogen receptor alpha rapidly activates the IGF-1 receptor pathway. J Biol
Chem 275, 18447-18453.
Kamei, Y., Xu, L., Heinzel, T., Torchia, J., Kurokawa, R., Gloss, B., Lin, S. C., Heyman,
R. A., Rose, D. W., Glass, C. K., and Rosenfeld, M. G. (1996). A CBP integrator
complex mediates transcriptional activation and AP-1 inhibition by nuclear
receptors. Cell 85, 403-414.
252
Kandouz, M., Siromachkova, M., Jacob, D., Chretien, Marquet B., Therwath, A., and
Gompel, A. (1996). Antagonism between estradiol and progestin on Bcl-2
expression in breast-cancer cells. Int J Cancer 68, 120-125.
Karnik, P. S., Kulkarni, S., Liu, X. P., Budd, G. T., and Bukowski, R. M. (1994).
Estrogen receptor mutations in tamoxifen-resistant breast cancer. Cancer Res 54,
349-353.
Kato, S., Endoh, H., Masuhiro, Y., Kitamoto, T., Uchiyama, S., Sasaki, H., Masushige,
S., Gotoh, Y., Nishida, E., Kawashima, H., and . (1995). Activation of the
estrogen receptor through phosphorylation by mitogen-activated protein kinase.
Science 270, 1491-1494.
Kelsey, J. L. and Berkowitz, G. S. (1988). Breast cancer epidemiology. Cancer Res 48,
5615-5623.
Kelsey, J. L. and Gammon, M. D. (1991). The epidemiology of breast cancer. CA Cancer
J Clin 41, 146-165.
Kenny, F. S., Hui, R., Musgrove, E. A., Gee, J. M., Blamey, R. W., Nicholson, R. I.,
Sutherland, R. L., and Robertson, J. F. (1999). Overexpression of cyclin D1
messenger RNA predicts for poor prognosis in estrogen receptor-positive breast
cancer. Clin Cancer Res 5, 2069-2076.
253
Kershah, S. M., Desouki, M. M., Koterba, K. L., and Rowan, B. G. (2004). Expression of
estrogen receptor coregulators in normal and malignant human endometrium.
Gynecol Oncol 92, 304-313.
Kim, H. P., Lee, J. Y., Jeong, J. K., Bae, S. W., Lee, H. K., and Jo, I. (1999).
Nongenomic stimulation of nitric oxide release by estrogen is mediated by
estrogen receptor alpha localized in caveolae. Biochem Biophys Res Commun
263, 257-262.
Kim, Y. J., Bjorklund, S., Li, Y., Sayre, M. H., and Kornberg, R. D. (1994). A
multiprotein mediator of transcriptional activation and its interaction with the Cterminal repeat domain of RNA polymerase II. Cell 20;77, 599-608.
Kirfel, J., Magin, T. M., and Reichelt, J. (2003). Keratins: a structural scaffold with
emerging functions. Cell Mol Life Sci 60, 56-71.
Klein-Hitpass, L., Schorpp, M., Wagner, U., and Ryffel, G. U. (1986). An estrogenresponsive element derived from the 5' flanking region of the Xenopus
vitellogenin A2 gene functions in transfected human cells. Cell 46, 1053-1061.
Kleinman, D., Karas, M., Danilenko, M., Arbell, A., Roberts, C. T., LeRoith, D., Levy,
J., and Sharoni, Y. (1996). Stimulation of endometrial cancer cell growth by
tamoxifen is associated with increased insulin-like growth factor (IGF)-I induced
tyrosine phosphorylation and reduction in IGF binding proteins. Endocrinology
137, 1089-1095.
254
Klinge, C. M. (2000). Estrogen receptor interaction with co-activators and co-repressors.
Steroids 65, 227-251.
Klotz, D. M., Hewitt, S. C., Ciana, P., Raviscioni, M., Lindzey, J. K., Foley, J., Maggi,
A., DiAugustine, R. P., and Korach, K. S. (2002). Requirement of estrogen
receptor-alpha in insulin-like growth factor-1 (IGF-1)-induced uterine responses
and in vivo evidence for IGF-1/estrogen receptor cross-talk. J Biol Chem 277,
8531-8537.
Krasilnikov, M. A. (2000). Phosphatidylinositol-3 kinase dependent pathways: the role in
control of cell growth, survival, and malignant transformation. Biochemistry
(Mosc ) 65, 59-67.
Kraus, W. L., McInerney, E. M., and Katzenellenbogen, B. S. (1995). Ligand-dependent,
transcriptionally productive association of the amino- and carboxyl-terminal
regions of a steroid hormone nuclear receptor. Proc Natl Acad Sci U S A 19;92,
12314-12318.
Krege, J. H., Hodgin, J. B., Couse, J. F., Enmark, E., Warner, M., Mahler, J. F., Sar, M.,
Korach, K. S., Gustafsson, J. A., and Smithies, O. (1998). Generation and
reproductive phenotypes of mice lacking estrogen receptor beta. Proc Natl Acad
Sci U S A 95, 15677-15682.
Kuang, S. Q., Liao, L., Zhang, H., Lee, A. V., O'Malley, B. W., and Xu, J. (2004).
AIB1/SRC-3 deficiency affects insulin-like growth factor I signaling pathway and
255
suppresses v-Ha-ras-induced breast cancer initiation and progression in mice.
Cancer Res 64, 1875-1885.
Kuiper, G. G., Enmark, E., Pelto-Huikko, M., Nilsson, S., and Gustafsson, J. A. (1996).
Cloning of a novel receptor expressed in rat prostate and ovary. Proc Natl Acad
Sci U S A 93, 5925-5930.
Kumar, V., Green, S., Stack, G., Berry, M., Jin, J. R., and Chambon, P. (1987).
Functional domains of the human estrogen receptor. Cell 51, 941-951.
Labrie, F., Labrie, C., Belanger, A., Simard, J., Giguere, V., Tremblay, A., and Tremblay,
G. (2001). EM-652 (SCH57068), a pure SERM having complete antiestrogenic
activity in the mammary gland and endometrium. J Steroid Biochem Mol Biol 79,
213-225.
Labrie, F., El Alfy, M., Berger, L., Labrie, C., Martel, C., Belanger, A., Candas, B., and
Pelletier, G. (2003). The combination of a novel selective estrogen receptor
modulator with an estrogen protects the mammary gland and uterus in a rodent
model: the future of postmenopausal women's health? Endocrinology 144, 47004706.
Lahooti, H., White, R., Danielian, P. S., and Parker, M. G. (1994). Characterization of
ligand-dependent phosphorylation of the estrogen receptor. Mol Endocrinol 8,
182-188.
Lannigan, D. A. (2003). Estrogen receptor phosphorylation. Steroids 68, 1-9.
256
Lanz, R. B., McKenna, N. J., Onate, S. A., Albrecht, U., Wong, J., Tsai, S. Y., Tsai, M.
J., and O'Malley, B. W. (1999). A steroid receptor coactivator, SRA, functions as
an RNA and is present in an SRC-1 complex. Cell 97, 17-27.
Lavinsky, R. M., Jepsen, K., Heinzel, T., Torchia, J., Mullen, T. M., Schiff, R., Del Rio,
A. L., Ricote, M., Ngo, S., Gemsch, J., Hilsenbeck, S. G., Osborne, C. K., Glass,
C. K., Rosenfeld, M. G., and Rose, D. W. (1998). Diverse signaling pathways
modulate nuclear receptor recruitment of N-CoR and SMRT complexes. Proc
Natl Acad Sci U S A 95, 2920-2925.
Le Goff, P., Montano, M. M., Schodin, D. J., and Katzenellenbogen, B. S. (1994).
Phosphorylation of the human estrogen receptor. Identification of hormoneregulated sites and examination of their influence on transcriptional activity. J
Biol Chem 269, 4458-4466.
Leake, R. (1996). 100 years of the endocrine battle against breast cancer. Lancet 347,
1780-1781.
Leclercq, G., Legros, N., and Piccart, M. J. (1992). Accumulation of a non-binding form
of estrogen receptor in MCF-7 cells under hydroxytamoxifen treatment. J Steroid
Biochem Mol Biol 41, 545-552.
Lee, H., Jiang, F., Wang, Q., Nicosia, S. V., Yang, J., Su, B., and Bai, W. (2000).
MEKK1 activation of human estrogen receptor alpha and stimulation of the
257
agonistic activity of 4-hydroxytamoxifen in endometrial and ovarian cancer cells.
Mol Endocrinol 14, 1882-1896.
Lee, H. and Bai, W. (2002). Regulation of estrogen receptor nuclear export by ligandinduced and p38-mediated receptor phosphorylation. Mol Cell Biol 22, 58355845.
Lee, K. C., Jessop, H., Suswillo, R., Zaman, G., and Lanyon, L. E. (2004). The adaptive
response of bone to mechanical loading in female transgenic mice is deficient in
the absence of oestrogen receptor-alpha and -beta. J Endocrinol 182, 193-201.
Leone, A., Flatow, U., King, C. R., Sandeen, M. A., Margulies, I. M., Liotta, L. A., and
Steeg, P. S. (1991). Reduced tumor incidence, metastatic potential, and cytokine
responsiveness of nm23-transfected melanoma cells. Cell 65, 25-35.
Leone, A., Flatow, U., VanHoutte, K., and Steeg, P. S. (1993). Transfection of human
nm23-H1 into the human MDA-MB-435 breast carcinoma cell line: effects on
tumor metastatic potential, colonization and enzymatic activity. Oncogene 8,
2325-2333.
Leung, B. S. and Potter, A. H. (1987). Mode of estrogen action on cell proliferative
kinetics in CAMA-1 cells. I. Effect of serum and estrogen. Cancer Invest 5, 187194.
Levin, E. R. (2003). Bidirectional signaling between the estrogen receptor and the
epidermal growth factor receptor. Mol Endocrinol 17, 309-317.
258
Leygue, E., Dotzlaw, H., Watson, P. H., and Murphy, L. C. (1998). Altered estrogen
receptor alpha and beta messenger RNA expression during human breast
tumorigenesis. Cancer Res 58, 3197-3201.
Li, H., Gomes, P. J., and Chen, J. D. (1997). RAC3, a steroid/nuclear receptor-associated
coactivator that is related to SRC-1 and TIF2. Proc Natl Acad Sci U S A 94,
8479-8484.
Lin, K. H., Wang, W. J., Wu, Y. H., and Cheng, S. Y. (2002). Activation of
antimetastatic Nm23-H1 gene expression by estrogen and its alpha-receptor.
Endocrinology 143, 467-475.
Lindner, D. J. and Borden, E. C. (1997). Synergistic antitumor effects of a combination
of interferon and tamoxifen on estrogen receptor-positive and receptor-negative
human tumor cell lines in vivo and in vitro. J Interferon Cytokine Res 17, 681693.
Liu, X. F. and Bagchi, M. K. (2004). Recruitment of Distinct Chromatin-modifying
Complexes by Tamoxifen-complexed Estrogen Receptor at Natural Target Gene
Promoters in Vivo. J Biol Chem 279, 15050-15058.
Liu, Y., el Ashry, D., Chen, D., Ding, I. Y., and Kern, F. G. (1995). MCF-7 breast cancer
cells overexpressing transfected c-erbB-2 have an in vitro growth advantage in
estrogen-depleted conditions and reduced estrogen-dependence and tamoxifensensitivity in vivo. Breast Cancer Res Treat 34, 97-117.
259
Liu, Z., Wong, J., Tsai, S. Y., Tsai, M. J., and O'Malley, B. W. (2001). Sequential
recruitment of steroid receptor coactivator-1 (SRC-1) and p300 enhances
progesterone receptor-dependent initiation and reinitiation of transcription from
chromatin. Proc Natl Acad Sci U S A 98, 12426-12431.
Lonard, D. M., Nawaz, Z., Smith, C. L., and O'Malley, B. W. (2000). The 26S
proteasome is required for estrogen receptor-alpha and coactivator turnover and
for efficient estrogen receptor-alpha transactivation. Mol Cell 5, 939-948.
Lopez, G. N., Turck, C. W., Schaufele, F., Stallcup, M. R., and Kushner, P. J. (2001).
Growth factors signal to steroid receptors through mitogen-activated protein
kinase regulation of p160 coactivator activity. J Biol Chem 276, 22177-22182.
Lu, J., Jiang, C., Kaeck, M., Ganther, H., Vadhanavikit, S., Ip, C., and Thompson, H.
(1995). Dissociation of the genotoxic and growth inhibitory effects of selenium.
Biochem Pharmacol 50, 213-219.
Lubahn, D. B., Moyer, J. S., Golding, T. S., Couse, J. F., Korach, K. S., and Smithies, O.
(1993). Alteration of reproductive function but not prenatal sexual development
after insertional disruption of the mouse estrogen receptor gene. Proc Natl Acad
Sci U S A 90, 11162-11166.
Luo, S., Sourla, A., Labrie, C., Gauthier, S., Merand, Y., Belanger, A., and Labrie, F.
(1998). Effect of twenty-four-week treatment with the antiestrogen EM-800 on
260
estrogen-sensitive parameters in intact and ovariectomized mice. Endocrinology
139, 2645-2656.
Maa, M. C., Leu, T. H., McCarley, D. J., Schatzman, R. C., and Parsons, S. J. (1995).
Potentiation of epidermal growth factor receptor-mediated oncogenesis by c-Src:
implications for the etiology of multiple human cancers. Proc Natl Acad Sci U S
A 92, 6981-6985.
Maciver, S. K. and Weeds, A. G. (1994). Actophorin preferentially binds monomeric
ADP-actin over ATP-bound actin: consequences for cell locomotion. FEBS Lett
347, 251-256.
Magnusson, C., Baron, J. A., Correia, N., Bergstrom, R., Adami, H. O., and Persson, I.
(1999). Breast-cancer risk following long-term oestrogen- and oestrogenprogestin-replacement therapy. Int J Cancer 81, 339-344.
Mallon, E., Osin, P., Nasiri, N., Blain, I., Howard, B., and Gusterson, B. (2000). The
basic pathology of human breast cancer. J Mammary Gland Biol Neoplasia 5,
139-163.
Mandlekar, S., Hebbar, V., Christov, K., and Kong, A. N. (2000). Pharmacodynamics of
tamoxifen and its 4-hydroxy and N-desmethyl metabolites: activation of caspases
and induction of apoptosis in rat mammary tumors and in human breast cancer
cell lines. Cancer Res 60, 6601-6606.
261
Mandlekar, S. and Kong, A. N. (2001). Mechanisms of tamoxifen-induced apoptosis.
Apoptosis 6, 469-477.
Mark, M., Yoshida-Komiya, H., Gehin, M., Liao, L., Tsai, M. J., O'Malley, B. W.,
Chambon, P., and Xu, J. (2004). Partially redundant functions of SRC-1 and TIF2
in postnatal survival and male reproduction. Proc Natl Acad Sci U S A 101, 44534458.
Marquez, D. C. and Pietras, R. J. (2001). Membrane-associated binding sites for estrogen
contribute to growth regulation of human breast cancer cells. Oncogene 20, 54205430.
Marshall, J. R. (2001). Larry Clark's legacy: randomized controlled, selenium-based
prostate cancer chemoprevention trials. Nutr Cancer 40, 74-77.
Martel, C., Labrie, C., Belanger, A., Gauthier, S., Merand, Y., Li, X., Provencher, L.,
Candas, B., and Labrie, F. (1998). Comparison of the effects of the new orally
active antiestrogen EM-800 with ICI 182 780 and toremifene on estrogensensitive parameters in the ovariectomized mouse. Endocrinology 139, 24862492.
Martel, C., Picard, S., Richard, V., Belanger, A., Labrie, C., and Labrie, F. (2000).
Prevention of bone loss by EM-800 and raloxifene in the ovariectomized rat. J
Steroid Biochem Mol Biol 74, 45-56.
262
Martin, M. B., Franke, T. F., Stoica, G. E., Chambon, P., Katzenellenbogen, B. S., Stoica,
B. A., McLemore, M. S., Olivo, S. E., and Stoica, A. (2000). A role for Akt in
mediating the estrogenic functions of epidermal growth factor and insulin-like
growth factor I. Endocrinology 141, 4503-4511.
Matunis, E. L., Matunis, M. J., and Dreyfuss, G. (1993). Association of individual
hnRNP proteins and snRNPs with nascent transcripts. J Cell Biol 121, 219-228.
Matunis, M. J., Xing, J., and Dreyfuss, G. (1994). The hnRNP F protein: unique primary
structure, nucleic acid-binding properties, and subcellular localization. Nucleic
Acids Res 22, 1059-1067.
Mayeda, A. and Krainer, A. R. (1992). Regulation of alternative pre-mRNA splicing by
hnRNP A1 and splicing factor SF2. Cell 68, 365-375.
McClelland, R. A., Gee, J. M., Francis, A. B., Robertson, J. F., Blamey, R. W., Wakeling,
A. E., and Nicholson, R. I. (1996). Short-term effects of pure anti-oestrogen ICI
182780 treatment on oestrogen receptor, epidermal growth factor receptor and
transforming growth factor-alpha protein expression in human breast cancer. Eur
J Cancer 32A, 413-416.
McKeage, K., Curran, M. P., and Plosker, G. L. (2004). Fulvestrant: a review of its use in
hormone receptor-positive metastatic breast cancer in postmenopausal women
with disease progression following antiestrogen therapy. Drugs 64, 633-648.
263
McKenna, N. J., Lanz, R. B., and O'Malley, B. W. (1999). Nuclear receptor coregulators:
cellular and molecular biology. Endocr Rev 20, 321-344.
McKenna, N. J. and O'Malley, B. W. (2000). From ligand to response: generating
diversity in nuclear receptor coregulator function. J Steroid Biochem Mol Biol 74,
351-356.
McPherson, K., Steel, C. M., and Dixon, J. M. (2000). ABC of breast diseases. Breast
cancer-epidemiology, risk factors, and genetics. BMJ 321, 624-628.
Medina, D. and Oborn, C. J. (1981). Differential effects of selenium on the growth of
mouse mammary cells in vitro. Cancer Lett 13, 333-344.
Medina, D., Thompson, H., Ganther, H., and Ip, C. (2001). Se-methylselenocysteine: a
new compound for chemoprevention of breast cancer. Nutr Cancer 40, 12-17.
Merrill, A. H., Jr., Henderson, J. M., Wang, E., McDonald, B. W., and Millikan, W. J.
(1984). Metabolism of vitamin B-6 by human liver. J Nutr 114, 1664-1674.
Michalides, R., Griekspoor, A., Balkenende, A., Verwoerd, D., Janssen, L., Jalink, K.,
Floore, A., Velds, A., van't Veer, L., and Neefjes, J. (2004). Tamoxifen resistance
by a conformational arrest of the estrogen receptor alpha after PKA activation in
breast cancer. Cancer Cell 5, 597-605.
264
Migliaccio, A., Di Domenico, M., Castoria, G., de Falco, A., Bontempo, P., Nola, E., and
Auricchio, F. (1996). Tyrosine kinase/p21ras/MAP-kinase pathway activation by
estradiol-receptor complex in MCF-7 cells. EMBO J 15, 1292-1300.
Migliaccio, A., Castoria, G., Di Domenico, M., de Falco, A., Bilancio, A., Lombardi, M.,
Barone, M. V., Ametrano, D., Zannini, M. S., Abbondanza, C., and Auricchio, F.
(2000). Steroid-induced androgen receptor-oestradiol receptor beta-Src complex
triggers prostate cancer cell proliferation. EMBO J 19, 5406-5417.
Mosselman, S., Polman, J., and Dijkema, R. (1996). ER beta: identification and
characterization of a novel human estrogen receptor. FEBS Lett 19;392, 49-53.
Mueller, S. O. and Korach, K. S. (2001). Estrogen receptors and endocrine diseases:
lessons from estrogen receptor knockout mice. Curr Opin Pharmacol 1, 613-619.
Murphy, L. C., Dotzlaw, H., Leygue, E., Coutts, A., and Watson, P. (1998). The
pathophysiological role of estrogen receptor variants in human breast cancer. J
Steroid Biochem Mol Biol 65, 175-180.
Nagaoka, R., Abe, H., Kusano, K., and Obinata, T. (1995). Concentration of cofilin, a
small actin-binding protein, at the cleavage furrow during cytokinesis. Cell Motil
Cytoskeleton 30, 1-7.
Nagy, L., Kao, H. Y., Chakravarti, D., Lin, R. J., Hassig, C. A., Ayer, D. E., Schreiber, S.
L., and Evans, R. M. (1997). Nuclear receptor repression mediated by a complex
containing SMRT, mSin3A, and histone deacetylase. Cell 89, 373-380.
265
Nakamori, S., Ishikawa, O., Ohigashi, H., Imaoka, S., Sasaki, Y., Kameyama, M.,
Kabuto, T., Furukawa, H., Iwanakga, T., and Kimura, N. (1993).
Clinicopathological features and prognostic significance of nucleoside
diphosphate kinase/nm23 gene product in human pancreatic exocrine neoplasms.
Int J Pancreatol 14, 125-133.
Nakielny, S. and Dreyfuss, G. (1997). Nuclear export of proteins and RNAs. Curr Opin
Cell Biol 9, 420-429.
Nawaz, Z., Lonard, D. M., Smith, C. L., Lev-Lehman, E., Tsai, S. Y., Tsai, M. J., and
O'Malley, B. W. (1999a). The Angelman syndrome-associated protein, E6-AP, is
a coactivator for the nuclear hormone receptor superfamily. Mol Cell Biol 19,
1182-1189.
Nawaz, Z., Lonard, D. M., Dennis, A. P., Smith, C. L., and O'Malley, B. W. (1999b).
Proteasome-dependent degradation of the human estrogen receptor. Proc Natl
Acad Sci U S A 96, 1858-1862.
Nelson, C. C., Hendy, S. C., Shukin, R. J., Cheng, H., Bruchovsky, N., Koop, B. F., and
Rennie, P. S. (1999a). Determinants of DNA sequence specificity of the
androgen, progesterone, and glucocorticoid receptors: evidence for differential
steroid receptor response elements. Mol Endocrinol 13, 2090-2107.
266
Nelson, K. G., Takahashi, T., Bossert, N. L., Walmer, D. K., and McLachlan, J. A.
(1991). Epidermal growth factor replaces estrogen in the stimulation of female
genital-tract growth and differentiation. Proc Natl Acad Sci U S A 88, 21-25.
Nelson, M. A., Porterfield, B. W., Jacobs, E. T., and Clark, L. C. (1999b). Selenium and
prostate cancer prevention. Semin Urol Oncol 17, 91-96.
Neuman, E., Ladha, M. H., Lin, N., Upton, T. M., Miller, S. J., DiRenzo, J., Pestell, R.
G., Hinds, P. W., Dowdy, S. F., Brown, M., and Ewen, M. E. (1997). Cyclin D1
stimulation of estrogen receptor transcriptional activity independent of cdk4. Mol
Cell Biol 17, 5338-5347.
Ngo, E. O., LePage, G. R., Thanassi, J. W., Meisler, N., and Nutter, L. M. (1998).
Absence of pyridoxine-5'-phosphate oxidase (PNPO) activity in neoplastic cells:
isolation, characterization, and expression of PNPO cDNA. Biochemistry 37,
7741-7748.
Ngwenya, S. and Safe, S. (2003). Cell context-dependent differences in the induction of
E2F-1 gene expression by 17 beta-estradiol in MCF-7 and ZR-75 cells.
Endocrinology 144, 1675-1685.
Nicholson, R. I., McClelland, R. A., Robertson, J. F., and Gee, J. M. (1999). Involvement
of steroid hormone and growth factor cross-talk in endocrine response in breast
cancer. Endocr Relat Cancer 6, 373-387.
267
Norfleet, A. M., Clarke, C. H., Gametchu, B., and Watson, C. S. (2000). Antibodies to
the estrogen receptor-alpha modulate rapid prolactin release from rat pituitary
tumor cells through plasma membrane estrogen receptors. FASEB J 14, 157-165.
Norris, J. D., Paige, L. A., Christensen, D. J., Chang, C. Y., Huacani, M. R., Fan, D.,
Hamilton, P. T., Fowlkes, D. M., and McDonnell, D. P. (1999). Peptide
antagonists of the human estrogen receptor. Science 285, 744-746.
Nuber, U., Schwarz, S., Kaiser, P., Schneider, R., and Scheffner, M. (1996). Cloning of
human ubiquitin-conjugating enzymes UbcH6 and UbcH7 (E2-F1) and
characterization of their interaction with E6-AP and RSP5. J Biol Chem 271,
2795-2800.
O'brien, S., Tefferi, A., and Valent, P. (2004). Chronic myelogenous leukemia and
myeloproliferative disease. Hematology (Am Soc Hematol Educ Program ) :14662.
Ogawa, S., Inoue, S., Watanabe, T., Hiroi, H., Orimo, A., Hosoi, T., Ouchi, Y., and
Muramatsu, M. (1998). The complete primary structure of human estrogen
receptor beta (hER beta) and its heterodimerization with ER alpha in vivo and in
vitro. Biochem Biophys Res Commun 243, 122-126.
Olayioye, M. A., Neve, R. M., Lane, H. A., and Hynes, N. E. (2000). The ErbB signaling
network: receptor heterodimerization in development and cancer. EMBO J 19,
3159-3167.
268
Onate, S. A., Tsai, S. Y., Tsai, M. J., and O'Malley, B. W. (1995). Sequence and
characterization of a coactivator for the steroid hormone receptor superfamily.
Science 270, 1354-1357.
Osborne, C. K. (1998). Tamoxifen in the treatment of breast cancer. N Engl J Med 339,
1609-1618.
Osborne, C. K., Bardou, V., Hopp, T. A., Chamness, G. C., Hilsenbeck, S. G., Fuqua, S.
A., Wong, J., Allred, D. C., Clark, G. M., and Schiff, R. (2003). Role of the
estrogen receptor coactivator AIB1 (SRC-3) and HER-2/neu in tamoxifen
resistance in breast cancer. J Natl Cancer Inst 95, 353-361.
Ostareck, D. H., Ostareck-Lederer, A., Wilm, M., Thiele, B. J., Mann, M., and Hentze,
M. W. (1997). mRNA silencing in erythroid differentiation: hnRNP K and hnRNP
E1 regulate 15-lipoxygenase translation from the 3' end. Cell 89, 597-606.
Overholser, J. P., Prewett, M. C., Hooper, A. T., Waksal, H. W., and Hicklin, D. J.
(2000). Epidermal growth factor receptor blockade by antibody IMC-C225
inhibits growth of a human pancreatic carcinoma xenograft in nude mice. Cancer
89, 74-82.
Paech, K., Webb, P., Kuiper, G. G., Nilsson, S., Gustafsson, J., Kushner, P. J., and
Scanlan, T. S. (1997). Differential ligand activation of estrogen receptors ERalpha
and ERbeta at AP1 sites. Science 277, 1508-1510.
269
Palmieri, C., Saji, S., Sakaguchi, H., Cheng, G., Sunters, A., O'Hare, M. J., Warner, M.,
Gustafsson, J. A., Coombes, R. C., and Lam, E. W. (2004). The expression of
oestrogen receptor (ER)-beta and its variants, but not ERalpha, in adult human
mammary fibroblasts. J Mol Endocrinol 33, 35-50.
Paramio, J. M. and Jorcano, J. L. (2002). Beyond structure: do intermediate filaments
modulate cell signalling Bioessays 24, 836-844.
Patrick, L. (2004). Selenium biochemistry and cancer: a review of the literature. Altern
Med Rev 9, 239-258.
Patterson, B. H. and Levander, O. A. (1997). Naturally occurring selenium compounds in
cancer chemoprevention trials: a workshop summary. Cancer Epidemiol
Biomarkers Prev 6, 63-69.
Perillo, B., Sasso, A., Abbondanza, C., and Palumbo, G. (2000). 17beta-estradiol inhibits
apoptosis in MCF-7 cells, inducing bcl-2 expression via two estrogen-responsive
elements present in the coding sequence. Mol Cell Biol 20, 2890-2901.
Pettersson, K., Grandien, K., Kuiper, G. G., and Gustafsson, J. A. (1997). Mouse
estrogen receptor beta forms estrogen response element-binding heterodimers
with estrogen receptor alpha. Mol Endocrinol 11, 1486-1496.
Picard, F., Gehin, M., Annicotte, J., Rocchi, S., Champy, M. F., O'Malley, B. W.,
Chambon, P., and Auwerx, J. (2002). SRC-1 and TIF2 control energy balance
between white and brown adipose tissues. Cell 111, 931-941.
270
Pietras, R. J., Arboleda, J., Reese, D. M., Wongvipat, N., Pegram, M. D., Ramos, L.,
Gorman, C. M., Parker, M. G., Sliwkowski, M. X., and Slamon, D. J. (1995).
HER-2 tyrosine kinase pathway targets estrogen receptor and promotes hormoneindependent growth in human breast cancer cells. Oncogene 10, 2435-2446.
Poirier, K. A. and Milner, J. A. (1983). Factors influencing the antitumorigenic properties
of selenium in mice. J Nutr 113, 2147-2154.
Porter, W., Saville, B., Hoivik, D., and Safe, S. (1997). Functional synergy between the
transcription factor Sp1 and the estrogen receptor. Mol Endocrinol 11, 1569-1580.
Potera, C., Rose, D. P., and Brown, R. R. (1977). Vitamin B6 deficiency in cancer
patients. Am J Clin Nutr 30, 1677-1679.
Powers, C. A., Mathur, M., Raaka, B. M., Ron, D., and Samuels, H. H. (1998). TLS
(translocated-in-liposarcoma) is a high-affinity interactor for steroid, thyroid
hormone, and retinoid receptors. Mol Endocrinol 12, 4-18.
Pratt, W. B. and Toft, D. O. (1997). Steroid receptor interactions with heat shock protein
and immunophilin chaperones. Endocr Rev 18, 306-360.
Pringa, E., Meier, I., Muller, U., Martinez-Noel, G., and Harbers, K. (2000). Disruption
of the gene encoding the ubiquitin-conjugating enzyme UbcM4 has no effect on
proliferation and in vitro differentiation of mouse embryonic stem cells. Biochim
Biophys Acta 1494, 75-82.
271
Pritchard, K. I., Paterson, A. H., Fine, S., Paul, N. A., Zee, B., Shepherd, L. E., AbuZahra, H., Ragaz, J., Knowling, M., Levine, M. N., Verma, S., Perrault, D.,
Walde, P. L., Bramwell, V. H., Poljicak, M., Boyd, N., Warr, D., Norris, B. D.,
Bowman, D., Armitage, G. R., Weizel, H., and Buckman, R. A. (1997).
Randomized trial of cyclophosphamide, methotrexate, and fluorouracil
chemotherapy added to tamoxifen as adjuvant therapy in postmenopausal women
with node-positive estrogen and/or progesterone receptor-positive breast cancer: a
report of the National Cancer Institute of Canada Clinical Trials Group. Breast
Cancer Site Group. J Clin Oncol 15, 2302-2311.
Qin, C., Singh, P., and Safe, S. (1999). Transcriptional activation of insulin-like growth
factor-binding protein-4 by 17beta-estradiol in MCF-7 cells: role of estrogen
receptor-Sp1 complexes. Endocrinology 140, 2501-2508.
Rabenoelina, F., Semlali, A., Duchesne, M. J., Freiss, G., Pons, M., and Badia, E. (2002).
Effect of prolonged hydroxytamoxifen treatment of MCF-7 cells on mitogen
activated kinase cascade. Int J Cancer 98, 698-706.
Rachez, C., Suldan, Z., Ward, J., Chang, C. P., Burakov, D., Erdjument-Bromage, H.,
Tempst, P., and Freedman, L. P. (1998). A novel protein complex that interacts
with the vitamin D3 receptor in a ligand-dependent manner and enhances VDR
transactivation in a cell-free system. Genes Dev 12, 1787-1800.
272
Ramachandran, C., Catelli, M. G., Schneider, W., and Shyamala, G. (1988). Estrogenic
regulation of uterine 90-kilodalton heat shock protein. Endocrinology 123, 956961.
Redman, C., Scott, J. A., Baines, A. T., Basye, J. L., Clark, L. C., Calley, C., Roe, D.,
Payne, C. M., and Nelson, M. A. (1998). Inhibitory effect of selenomethionine on
the growth of three selected human tumor cell lines. Cancer Lett 125, 103-110.
Reissig, D., Clement, J., Sanger, J., Berndt, A., Kosmehl, H., and Bohmer, F. D. (2001).
Elevated activity and expression of Src-family kinases in human breast carcinoma
tissue versus matched non-tumor tissue. J Cancer Res Clin Oncol 127, 226-230.
Repp, R., van Ojik, H. H., Valerius, T., Groenewegen, G., Wieland, G., Oetzel, C.,
Stockmeyer, B., Becker, W., Eisenhut, M., Steininger, H., Deo, Y. M., Blijham,
G. H., Kalden, J. R., van de Winkel, J. G., and Gramatzki, M. (2003). Phase I
clinical trial of the bispecific antibody MDX-H210 (anti-FcgammaRI x anti-HER2/neu) in combination with Filgrastim (G-CSF) for treatment of advanced breast
cancer. Br J Cancer 89, 2234-2243.
Rowan, B. G., Garrison, N., Weigel, N. L., and O'Malley, B. W. (2000a). 8-Bromo-cyclic
AMP induces phosphorylation of two sites in SRC-1 that facilitate ligandindependent activation of the chicken progesterone receptor and are critical for
functional cooperation between SRC-1 and CREB binding protein. Mol Cell Biol
20, 8720-8730.
273
Rowan, B. G., Weigel, N. L., and O'Malley, B. W. (2000b). Phosphorylation of steroid
receptor coactivator-1. Identification of the phosphorylation sites and
phosphorylation through the mitogen-activated protein kinase pathway. J Biol
Chem 275, 4475-4483.
Russo, J., Tay, L. K., and Russo, I. H. (1982). Differentiation of the mammary gland and
susceptibility to carcinogenesis. Breast Cancer Res Treat 2, 5-73.
Ryan (1999). Ryan: Kistner's Gynecology & Women's Health, 7th ed.
Sakamoto, T., Eguchi, H., Omoto, Y., Ayabe, T., Mori, H., and Hayashi, S. (2002).
Estrogen receptor-mediated effects of tamoxifen on human endometrial cancer
cells. Mol Cell Endocrinol 192, 93-104.
Samudio, I., Vyhlidal, C., Wang, F., Stoner, M., Chen, I., Kladde, M., Barhoumi, R.,
Burghardt, R., and Safe, S. (2001). Transcriptional activation of deoxyribonucleic
acid polymerase alpha gene expression in MCF-7 cells by 17 beta-estradiol.
Endocrinology 142, 1000-1008.
Sar, M. and Welsch, F. (1999). Differential expression of estrogen receptor-beta and
estrogen receptor-alpha in the rat ovary. Endocrinology 140, 963-971.
Sarkar, S., Biswas, S. C., Chatterjee, O., and Sarkar, P. K. (1999). Protein kinase A
linked phosphorylation mediates triiodothyronine induced actin gene expression
in developing brain. Brain Res Mol Brain Res 67, 158-164.
274
Schiff, R., Massarweh, S., Shou, J., and Osborne, C. K. (2003). Breast cancer endocrine
resistance: how growth factor signaling and estrogen receptor coregulators
modulate response. Clin Cancer Res 9, 447S-454S.
Schlaepfer, D. D., Jones, K. C., and Hunter, T. (1998). Multiple Grb2-mediated integrinstimulated signaling pathways to ERK2/mitogen-activated protein kinase:
summation of both c-Src- and focal adhesion kinase-initiated tyrosine
phosphorylation events. Mol Cell Biol 18, 2571-2585.
Schomberg, D. W., Couse, J. F., Mukherjee, A., Lubahn, D. B., Sar, M., Mayo, K. E., and
Korach, K. S. (1999). Targeted disruption of the estrogen receptor-alpha gene in
female mice: characterization of ovarian responses and phenotype in the adult.
Endocrinology 140, 2733-2744.
Shah, Y. M., Basrur, V., and Rowan, B. G. (2004). Selective estrogen receptor modulator
regulated proteins in endometrial cancer cells. Mol Cell Endocrinol 219, 127-139.
Shang, Y., Hu, X., DiRenzo, J., Lazar, M. A., and Brown, M. (2000). Cofactor dynamics
and sufficiency in estrogen receptor-regulated transcription. Cell 103, 843-852.
Shang, Y. and Brown, M. (2002). Molecular determinants for the tissue specificity of
SERMs. Science 295, 2465-2468.
Shibata, H., Spencer, T. E., Onate, S. A., Jenster, G., Tsai, S. Y., Tsai, M. J., and
O'Malley, B. W. (1997). Role of co-activators and co-repressors in the mechanism
275
of steroid/thyroid receptor action. Recent Prog Horm Res 52:141-64; discussion
164-5., 141-164.
Shoker, B. S., Jarvis, C., Sibson, D. R., Walker, C., and Sloane, J. P. (1999a). Oestrogen
receptor expression in the normal and pre-cancerous breast. J Pathol 188, 237244.
Shoker, B. S., Jarvis, C., Clarke, R. B., Anderson, E., Hewlett, J., Davies, M. P., Sibson,
D. R., and Sloane, J. P. (1999b). Estrogen receptor-positive proliferating cells in
the normal and precancerous breast. Am J Pathol 155, 1811-1815.
Shyamala, G., Gauthier, Y., Moore, S. K., Catelli, M. G., and Ullrich, S. J. (1989).
Estrogenic regulation of murine uterine 90-kilodalton heat shock protein gene
expression. Mol Cell Biol 9, 3567-3570.
Simard, J., Labrie, C., Belanger, A., Gauthier, S., Singh, S. M., Merand, Y., and Labrie,
F. (1997). Characterization of the effects of the novel non-steroidal antiestrogen
EM-800 on basal and estrogen-induced proliferation of T-47D, ZR-75-1 and
MCF-7 human breast cancer cells in vitro. Int J Cancer 73, 104-112.
Sinha, R., Said, T. K., and Medina, D. (1996). Organic and inorganic selenium
compounds inhibit mouse mammary cell growth in vitro by different cellular
pathways. Cancer Lett 107, 277-284.
276
Sinha, R. and Medina, D. (1997). Inhibition of cdk2 kinase activity by
methylselenocysteine in synchronized mouse mammary epithelial tumor cells.
Carcinogenesis 18, 1541-1547.
Sinha, R., Kiley, S. C., Lu, J. X., Thompson, H. J., Moraes, R., Jaken, S., and Medina, D.
(1999). Effects of methylselenocysteine on PKC activity, cdk2 phosphorylation
and gadd gene expression in synchronized mouse mammary epithelial tumor
cells. Cancer Lett 146, 135-145.
Sinha, R., Unni, E., Ganther, H. E., and Medina, D. (2001). Methylseleninic acid, a
potent growth inhibitor of synchronized mouse mammary epithelial tumor cells in
vitro. Biochem Pharmacol 61, 311-317.
Sivaraman, L., Nawaz, Z., Medina, D., Conneely, O. M., and O'Malley, B. W. (2000).
The dual function steroid receptor coactivator/ubiquitin protein-ligase integrator
E6-AP is overexpressed in mouse mammary tumorigenesis. Breast Cancer Res
Treat 62, 185-195.
Slamon, D. J., Leyland-Jones, B., Shak, S., Fuchs, H., Paton, V., Bajamonde, A.,
Fleming, T., Eiermann, W., Wolter, J., Pegram, M., Baselga, J., and Norton, L.
(2001). Use of chemotherapy plus a monoclonal antibody against HER2 for
metastatic breast cancer that overexpresses HER2. N Engl J Med 344, 783-792.
277
Smith, C. L., Conneely, O. M., and O'Malley, B. W. (1993). Modulation of the ligandindependent activation of the human estrogen receptor by hormone and
antihormone. Proc Natl Acad Sci U S A 90, 6120-6124.
Smith, C. L., Nawaz, Z., and O'Malley, B. W. (1997). Coactivator and corepressor
regulation of the agonist/antagonist activity of the mixed antiestrogen, 4hydroxytamoxifen. Mol Endocrinol 11, 657-666.
Smith, C. L., DeVera, D. G., Lamb, D. J., Nawaz, Z., Jiang, Y. H., Beaudet, A. L., and
O'Malley, B. W. (2002). Genetic ablation of the steroid receptor coactivatorubiquitin ligase, E6-AP, results in tissue-selective steroid hormone resistance and
defects in reproduction. Mol Cell Biol 22, 525-535.
Sourla, A., Luo, S., Labrie, C., Belanger, A., and Labrie, F. (1997). Morphological
changes induced by 6-month treatment of intact and ovariectomized mice with
tamoxifen and the pure antiestrogen EM-800. Endocrinology 138, 5605-5617.
Speirs, V., Carder, P. J., Lane, S., Dodwell, D., Lansdown, M. R., and Hanby, A. M.
(2004). Oestrogen receptor beta: what it means for patients with breast cancer.
Lancet Oncol 5, 174-181.
Spencer, T. E., Jenster, G., Burcin, M. M., Allis, C. D., Zhou, J., Mizzen, C. A.,
McKenna, N. J., Onate, S. A., Tsai, S. Y., Tsai, M. J., and O'Malley, B. W.
(1997). Steroid receptor coactivator-1 is a histone acetyltransferase. Nature 389,
194-198.
278
Sridhar, S. S. and Shepherd, F. A. (2003a). Targeting angiogenesis: a review of
angiogenesis inhibitors in the treatment of lung cancer. Lung Cancer 42 Suppl
1:S81-91.
Sridhar, S. S., Seymour, L., and Shepherd, F. A. (2003b). Inhibitors of epidermal-growthfactor receptors: a review of clinical research with a focus on non-small-cell lung
cancer. Lancet Oncol 4, 397-406.
Stack, G., Kumar, V., Green, S., Ponglikitmongkol, M., Berry, M., Rio, M. C., Nunez, A.
M., Roberts, M., Koehl, C., Bellocq, P., and . (1988). Structure and function of
the pS2 gene and estrogen receptor in human breast cancer cells. Cancer Treat
Res 40, 185-206.
Stallard, S., Hole, D. A., Purushotham, A. D., Hiew, L. Y., Mehanna, H., Cordiner, C.,
Dobson, H., Mallon, E. A., and George, W. D. (2001). Ductal carcinoma in situ of
the breast -- among factors predicting for recurrence, distance from the nipple is
important. Eur J Surg Oncol 27, 373-377.
Steeg, P. S., Bevilacqua, G., Kopper, L., Thorgeirsson, U. P., Talmadge, J. E., Liotta, L.
A., and Sobel, M. E. (1988). Evidence for a novel gene associated with low tumor
metastatic potential. J Natl Cancer Inst 80, 200-204.
Stefano, G. B., Cadet, P., Breton, C., Goumon, Y., Prevot, V., Dessaint, J. P.,
Beauvillain, J. C., Roumier, A. S., Welters, I., and Salzet, M. (2000a). Estradiolstimulated nitric oxide release in human granulocytes is dependent on intracellular
279
calcium transients: evidence of a cell surface estrogen receptor. Blood 95, 39513958.
Stefano, G. B., Prevot, V., Beauvillain, J. C., Cadet, P., Fimiani, C., Welters, I.,
Fricchione, G. L., Breton, C., Lassalle, P., Salzet, M., and Bilfinger, T. V.
(2000b). Cell-surface estrogen receptors mediate calcium-dependent nitric oxide
release in human endothelia. Circulation 101, 1594-1597.
Steinmetz, A. C., Renaud, J. P., and Moras, D. (2001). Binding of ligands and activation
of transcription by nuclear receptors. Annu Rev Biophys Biomol Struct 30, 32959.
Stendahl, M., Kronblad, A., Ryden, L., Emdin, S., Bengtsson, N. O., and Landberg, G.
(2004). Cyclin D1 overexpression is a negative predictive factor for tamoxifen
response in postmenopausal breast cancer patients. Br J Cancer 90, 1942-1948.
Stoica, A., Pentecost, E., and Martin, M. B. (2000). Effects of selenite on estrogen
receptor-alpha expression and activity in MCF-7 breast cancer cells. J Cell
Biochem 79, 282-292.
Sun, M., Paciga, J. E., Feldman, R. I., Yuan, Z., Coppola, D., Lu, Y. Y., Shelley, S. A.,
Nicosia, S. V., and Cheng, J. Q. (2001). Phosphatidylinositol-3-OH Kinase
(PI3K)/AKT2, activated in breast cancer, regulates and is induced by estrogen
receptor alpha (ERalpha) via interaction between ERalpha and PI3K. Cancer Res
61, 5985-5991.
280
Sutherland, R. L., Hall, R. E., and Taylor, I. W. (1983). Cell proliferation kinetics of
MCF-7 human mammary carcinoma cells in culture and effects of tamoxifen on
exponentially growing and plateau-phase cells. Cancer Res 43, 3998-4006.
Swain, S. M. (1989a). Noninvasive breast cancer: Part 2. Lobular carcinoma in situ-incidence, presentation, guidelines to treatment. Oncology (Huntingt) 3, 35-40.
Swain, S. M. (1989b). Noninvasive breast cancer: Part 1. Ductal carcinoma in situ-incidence, presentation, guidelines to treatment. Oncology (Huntingt) 3, 25-31.
Swede, H., Dong, Y., Reid, M., Marshall, J., and Ip, C. (2003). Cell cycle arrest
biomarkers in human lung cancer cells after treatment with selenium in culture.
Cancer Epidemiol Biomarkers Prev 12, 1248-1252.
Takeshita, A., Yen, P. M., Misiti, S., Cardona, G. R., Liu, Y., and Chin, W. W. (1996).
Molecular cloning and properties of a full-length putative thyroid hormone
receptor coactivator. Endocrinology 137, 3594-3597.
Tang, P. Z., Gannon, M. J., Andrew, A., and Miller, D. (1995). Evidence for oestrogenic
regulation of heat shock protein expression in human endometrium and steroidresponsive cell lines. Eur J Endocrinol 133, 598-605.
Terenius, L. (1971). Structure-activity relationships of anti-oestrogens with regard to
interaction with 17-beta-oestradiol in the mouse uterus and vagina. Acta
Endocrinol (Copenh) 66, 431-447.
281
Thomas, S. M. and Brugge, J. S. (1997). Cellular functions regulated by Src family
kinases. Annu Rev Cell Dev Biol 13, 513-609.
Tora, L., White, J., Brou, C., Tasset, D., Webster, N., Scheer, E., and Chambon, P.
(1989). The human estrogen receptor has two independent nonacidic
transcriptional activation functions. Cell 59, 477-487.
Torchia, J., Rose, D. W., Inostroza, J., Kamei, Y., Westin, S., Glass, C. K., and
Rosenfeld, M. G. (1997). The transcriptional co-activator p/CIP binds CBP and
mediates nuclear-receptor function. Nature 387, 677-684.
Traxler, P., Bold, G., Buchdunger, E., Caravatti, G., Furet, P., Manley, P., O'Reilly, T.,
Wood, J., and Zimmermann, J. (2001). Tyrosine kinase inhibitors: from rational
design to clinical trials. Med Res Rev 21, 499-512.
Tremblay, A., Tremblay, G. B., Labrie, C., Labrie, F., and Giguere, V. (1998). EM-800, a
novel antiestrogen, acts as a pure antagonist of the transcriptional functions of
estrogen receptors alpha and beta. Endocrinology 139, 111-118.
Tremblay, A., Tremblay, G. B., Labrie, F., and Giguere, V. (1999). Ligand-independent
recruitment of SRC-1 to estrogen receptor beta through phosphorylation of
activation function AF-1. Mol Cell 3, 513-519.
Tremblay, G. B., Tremblay, A., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., Labrie, F.,
and Giguere, V. (1997). Cloning, chromosomal localization, and functional
analysis of the murine estrogen receptor beta. Mol Endocrinol 11, 353-365.
282
Tsai, M. J. and O'Malley, B. W. (1994). Molecular mechanisms of action of
steroid/thyroid receptor superfamily members. Annu Rev Biochem 63, 451-86.
Twal, W. O., Czirok, A., Hegedus, B., Knaak, C., Chintalapudi, M. R., Okagawa, H.,
Sugi, Y., and Argraves, W. S. (2001). Fibulin-1 suppression of fibronectinregulated cell adhesion and motility. J Cell Sci 114, 4587-4598.
Tzeng, D. Z. and Klinge, C. M. (1996). Phosphorylation of purified estradiol-liganded
estrogen receptor by casein kinase II increases estrogen response element binding
but does not alter ligand stability. Biochem Biophys Res Commun 223, 554-560.
Tzukerman, M. T., Esty, A., Santiso-Mere, D., Danielian, P., Parker, M. G., Stein, R. B.,
Pike, J. W., and McDonnell, D. P. (1994). Human estrogen receptor
transactivational capacity is determined by both cellular and promoter context and
mediated by two functionally distinct intramolecular regions. Mol Endocrinol 8,
21-30.
Vidal, O., Kindblom, L. G., and Ohlsson, C. (1999). Expression and localization of
estrogen receptor-beta in murine and human bone. J Bone Miner Res 14, 923-929.
Vladusic, E. A., Hornby, A. E., Guerra-Vladusic, F. K., Lakins, J., and Lupu, R. (2000).
Expression and regulation of estrogen receptor beta in human breast tumors and
cell lines. Oncol Rep 7, 157-167.
283
Voegel, J. J., Heine, M. J., Zechel, C., Chambon, P., and Gronemeyer, H. (1996). TIF2, a
160 kDa transcriptional mediator for the ligand-dependent activation function AF2 of nuclear receptors. EMBO J 15, 3667-3675.
Vogel, C. L., Cobleigh, M. A., Tripathy, D., Gutheil, J. C., Harris, L. N., Fehrenbacher,
L., Slamon, D. J., Murphy, M., Novotny, W. F., Burchmore, M., Shak, S.,
Stewart, S. J., and Press, M. (2002). Efficacy and safety of trastuzumab as a single
agent in first-line treatment of HER2-overexpressing metastatic breast cancer. J
Clin Oncol 20, 719-726.
Wagner, B. L., Norris, J. D., Knotts, T. A., Weigel, N. L., and McDonnell, D. P. (1998).
The nuclear corepressors NCoR and SMRT are key regulators of both ligand- and
8-bromo-cyclic AMP-dependent transcriptional activity of the human
progesterone receptor. Mol Cell Biol 18, 1369-1378.
Wakeling, A. E., Dukes, M., and Bowler, J. (1991). A potent specific pure antiestrogen
with clinical potential. Cancer Res 51, 3867-3873.
Walinder, O. (1968). Identification of a phosphate-incorporating protein from bovine
liver as nucleoside diphosphate kinase and isolation of 1-32P-phosphohistidine, 332P-phosphohistidine, and N-epsilon-32P-phospholysine from erythrocytic
nucleoside diphosphate kinase, incubated with adenosine triphosphate-32P. J Biol
Chem 243, 3947-3952.
284
Wallace, P. K., Romet-Lemonne, J. L., Chokri, M., Kasper, L. H., Fanger, M. W., and
Fadul, C. E. (2000). Production of macrophage-activated killer cells for targeting
of glioblastoma cells with bispecific antibody to FcgammaRI and the epidermal
growth factor receptor. Cancer Immunol Immunother 49, 493-503.
Walter, S. and Buchner, J. (2002). Molecular chaperones--cellular machines for protein
folding. Angew Chem Int Ed Engl 41, 1098-1113.
Wang, F., Samudio, I., and Safe, S. (2001a). Transcriptional activation of rat creatine
kinase B by 17beta-estradiol in MCF-7 cells involves an estrogen responsive
element and GC-rich sites. J Cell Biochem 84, 156-172.
Wang, H., Huang, Z. Q., Xia, L., Feng, Q., Erdjument-Bromage, H., Strahl, B. D.,
Briggs, S. D., Allis, C. D., Wong, J., Tempst, P., and Zhang, Y. (2001b).
Methylation of histone H4 at arginine 3 facilitating transcriptional activation by
nuclear hormone receptor. Science 293, 853-857.
Wang, R. A., Mazumdar, A., Vadlamudi, R. K., and Kumar, R. (2002). P21-activated
kinase-1 phosphorylates and transactivates estrogen receptor-alpha and promotes
hyperplasia in mammary epithelium. EMBO J 21, 5437-5447.
Wang, Z., Rose, D. W., Hermanson, O., Liu, F., Herman, T., Wu, W., Szeto, D.,
Gleiberman, A., Krones, A., Pratt, K., Rosenfeld, R., Glass, C. K., and Rosenfeld,
M. G. (2000). Regulation of somatic growth by the p160 coactivator p/CIP. Proc
Natl Acad Sci U S A 97, 13549-13554.
285
Wang, Z., Jiang, C., Ganther, H., and Lu, J. (2001c). Antimitogenic and proapoptotic
activities of methylseleninic acid in vascular endothelial cells and associated
effects on PI3K-AKT, ERK, JNK and p38 MAPK signaling. Cancer Res 61,
7171-7178.
Wang, Z., Jiang, C., and Lu, J. (2002). Induction of caspase-mediated apoptosis and cellcycle G1 arrest by selenium metabolite methylselenol. Mol Carcinog 34, 113-120.
Washburn, T., Hocutt, A., Brautigan, D. L., and Korach, K. S. (1991). Uterine estrogen
receptor in vivo: phosphorylation of nuclear specific forms on serine residues.
Mol Endocrinol 5, 235-242.
Watrach, A. M., Milner, J. A., and Watrach, M. A. (1982). Effect of selenium on growth
rate of canine mammary carcinoma cells in athymic nude mice. Cancer Lett 15,
137-143.
Watrach, A. M., Milner, J. A., Watrach, M. A., and Poirier, K. A. (1984). Inhibition of
human breast cancer cells by selenium. Cancer Lett 25, 41-47.
Watson, C. S., Norfleet, A. M., Pappas, T. C., and Gametchu, B. (1999). Rapid actions of
estrogens in GH3/B6 pituitary tumor cells via a plasma membrane version of
estrogen receptor-alpha. Steroids 64, 5-13.
Watts, N. B. (1999). Postmenopausal osteoporosis. Obstet Gynecol Surv 54, 532-538.
286
Weatherman, R. V., Fletterick, R. J., and Scanlan, T. S. (1999). Nuclear-receptor ligands
and ligand-binding domains. Annu Rev Biochem 68, 559-81.
Webb, P., Lopez, G. N., Uht, R. M., and Kushner, P. J. (1995). Tamoxifen activation of
the estrogen receptor/AP-1 pathway: potential origin for the cell-specific
estrogen-like effects of antiestrogens. Mol Endocrinol 9, 443-456.
Webb, P., Nguyen, P., Shinsako, J., Anderson, C., Feng, W., Nguyen, M. P., Chen, D.,
Huang, S. M., Subramanian, S., McKinerney, E., Katzenellenbogen, B. S.,
Stallcup, M. R., and Kushner, P. J. (1998). Estrogen receptor activation function 1
works by binding p160 coactivator proteins. Mol Endocrinol 12, 1605-1618.
Webb, P., Nguyen, P., Valentine, C., Lopez, G. N., Kwok, G. R., McInerney, E.,
Katzenellenbogen, B. S., Enmark, E., Gustafsson, J. A., Nilsson, S., and Kushner,
P. J. (1999). The estrogen receptor enhances AP-1 activity by two distinct
mechanisms with different requirements for receptor transactivation functions.
Mol Endocrinol 13, 1672-1685.
Weigel, N. L. and Zhang, Y. (1998). Ligand-independent activation of steroid hormone
receptors. J Mol Med 76, 469-479.
Weis, K. E., Ekena, K., Thomas, J. A., Lazennec, G., and Katzenellenbogen, B. S.
(1996). Constitutively active human estrogen receptors containing amino acid
substitutions for tyrosine 537 in the receptor protein. Mol Endocrinol 10, 13881398.
287
Weissman, A. M. (2001). Themes and variations on ubiquitylation. Nat Rev Mol Cell
Biol 2, 169-178.
Weniger, J. P. (1990). Aromatase activity in fetal gonads of mammals. J Dev Physiol 14,
303-306.
Wilcken, N. R., Prall, O. W., Musgrove, E. A., and Sutherland, R. L. (1997). Inducible
overexpression of cyclin D1 in breast cancer cells reverses the growth-inhibitory
effects of antiestrogens. Clin Cancer Res 3, 849-854.
Willems, R., Slegers, H., Rodrigus, I., Moulijn, A. C., Lenjou, M., Nijs, G., Berneman, Z.
N., and Van Bockstaele, D. R. (2002). Extracellular nucleoside diphosphate
kinase NM23/NDPK modulates normal hematopoietic differentiation. Exp
Hematol 30, 640-648.
Wise, P. M., Dubal, D. B., Wilson, M. E., Rau, S. W., and Liu, Y. (2001). Estrogens:
trophic and protective factors in the adult brain. Front Neuroendocrinol 22, 33-66.
Won, J., Yim, J., and Kim, T. K. (2002). Sp1 and Sp3 recruit histone deacetylase to
repress transcription of human telomerase reverse transcriptase (hTERT)
promoter in normal human somatic cells. J Biol Chem 277, 38230-38238.
Wu, K., Helzlsouer, K. J., Comstock, G. W., Hoffman, S. C., Nadeau, M. R., and Selhub,
J. (1999). A prospective study on folate, B12, and pyridoxal 5'-phosphate (B6)
and breast cancer. Cancer Epidemiol Biomarkers Prev 8, 209-217.
288
Wu, R. C., Qin, J., Yi, P., Wong, J., Tsai, S. Y., Tsai, M. J., and O'Malley, B. W. (2004).
Selective phosphorylations of the SRC-3/AIB1 coactivator integrate genomic
reponses to multiple cellular signaling pathways. Mol Cell 15, 937-949.
Wu, W. X., Derks, J. B., Zhang, Q., and Nathanielsz, P. W. (1996). Changes in heat
shock protein-90 and -70 messenger ribonucleic acid in uterine tissues of the ewe
in relation to parturition and regulation by estradiol and progesterone.
Endocrinology 137, 5685-5693.
Xie, W., Duan, R., and Safe, S. (1999). Estrogen induces adenosine deaminase gene
expression in MCF-7 human breast cancer cells: role of estrogen receptor-Sp1
interactions. Endocrinology 140, 219-227.
Xu, J., Qiu, Y., DeMayo, F. J., Tsai, S. Y., Tsai, M. J., and O'Malley, B. W. (1998).
Partial hormone resistance in mice with disruption of the steroid receptor
coactivator-1 (SRC-1) gene. Science 20;279, 1922-1925.
Xu, J., Liao, L., Ning, G., Yoshida-Komiya, H., Deng, C., and O'Malley, B. W. (2000).
The steroid receptor coactivator SRC-3 (p/CIP/RAC3/AIB1/ACTR/TRAM-1) is
required for normal growth, puberty, female reproductive function, and mammary
gland development. Proc Natl Acad Sci U S A 97, 6379-6384.
Yan, F., Gao, X., Lonard, D. M., and Nawaz, Z. (2003). Specific ubiquitin-conjugating
enzymes promote degradation of specific nuclear receptor coactivators. Mol
Endocrinol 17, 1315-1331.
289
Yan, L., Boylan, L. M., and Spallholz, J. E. (1991). Effect of dietary selenium and
magnesium on human mammary tumor growth in athymic nude mice. Nutr
Cancer 16, 239-248.
Yang, X. D., Jia, X. C., Corvalan, J. R., Wang, P., and Davis, C. G. (2001). Development
of ABX-EGF, a fully human anti-EGF receptor monoclonal antibody, for cancer
therapy. Crit Rev Oncol Hematol 38, 17-23.
Yarden, Y. and Sliwkowski, M. X. (2001). Untangling the ErbB signalling network. Nat
Rev Mol Cell Biol 2, 127-137.
Yokouchi, M., Kondo, T., Houghton, A., Bartkiewicz, M., Horne, W. C., Zhang, H.,
Yoshimura, A., and Baron, R. (1999). Ligand-induced ubiquitination of the
epidermal growth factor receptor involves the interaction of the c-Cbl RING
finger and UbcH7. J Biol Chem 274, 31707-31712.
Yokouchi, M., Kondo, T., Sanjay, A., Houghton, A., Yoshimura, A., Komiya, S., Zhang,
H., and Baron, R. (2001). Src-catalyzed phosphorylation of c-Cbl leads to the
interdependent ubiquitination of both proteins. J Biol Chem 276, 35185-35193.
Zhang, S. M., Willett, W. C., Selhub, J., Hunter, D. J., Giovannucci, E. L., Holmes, M.
D., Colditz, G. A., and Hankinson, S. E. (2003). Plasma folate, vitamin B6,
vitamin B12, homocysteine, and risk of breast cancer. J Natl Cancer Inst 95, 373380.
290
Zhu, Z., Jiang, W., Ganther, H. E., and Thompson, H. J. (2002). Mechanisms of cell
cycle arrest by methylseleninic acid. Cancer Res 62, 156-164.
Zu, K. and Ip, C. (2003). Synergy between selenium and vitamin E in apoptosis induction
is associated with activation of distinctive initiator caspases in human prostate
cancer cells. Cancer Res 63, 6988-6995.
Zugmaier, G., Ennis, B. W., Deschauer, B., Katz, D., Knabbe, C., Wilding, G., Daly, P.,
Lippman, M. E., and Dickson, R. B. (1989). Transforming growth factors type
beta 1 and beta 2 are equipotent growth inhibitors of human breast cancer cell
lines. J Cell Physiol 141, 353-361.
291
ABSTRACT
Tamoxifen is the most widely prescribed selective estrogen receptor modulator
(SERM) for breast cancer. Despite the benefits of tamoxifen therapy, almost all
tamoxifen-responsive breast cancer patients develop resistance to therapy and tamoxifen
exhibits ER agonist action in the uterus that is associated with an increased incidence of
endometrial cancer. These problems with tamoxifen therapy indicate a need to identify
new biomarkers for tamoxifen therapy that are predictive of tamoxifen resistance and/or
the agonist effects associated in the endometrium. Protein expression profiles associated
with a graded estrogen response were generated in an ER-positive and tamoxifen resistant
Ishikawa endometrial adenocarcinoma cell-line using two-dimensional gel
electrophoresis and mass spectrometry sequencing. Among the identified proteins were
chaperones proteins, RNA and RNA polymerase binding proteins, cytoskeletal proteins,
an ubiquitin ligase, and a multi-functional kinase. The proteins identified may serve as
useful biomarkers for development of tamoxifen resistance or serve as novel tumor
targets.
In related projects designed to identify novel mechanisms to increase tamoxifen
efficacy, it was found that src kinase inhibitors and the micronutrient selenium modulated
ERα signaling and potentiated the antagonist action of tamoxifen in sensitive and
resistant breast and endometrial cell lines through discrete mechanisms. Src kinase
induced ERα phosphorylation at serine 167 via the PI3K/AKT pathway that was shown
to be critical for ERα promoter interaction. In addition, src specifically enhanced the
292
transcriptional activity of steroid receptor coactivator-1 (SRC-1), which was sufficient to
enhance its coactivation of ERα. Inhibition of src kinase blocked these mechanisms and
prevented tamoxifen agonist action in tamoxifen resistant cell lines.
Methylseleninic acid (MSA) is a monomethylated selenium compound that
inhibits growth of cancer cells in vitro. MSA decreased ERα mRNA and protein levels
in tamoxifen-sensitive and -resistant breast and endometrial cancer cell lines in vitro that
resulted in subsequent antagonism of estradiol-dependent ERE2e1b-luciferase and
endogenous c-myc and pS2 gene expression. MSA also reversed tamoxifen activation of
these genes in endometrial Ishikawa and HEC-1A cells. In addition, MSA potentiated
the growth inhibitory affects of tamoxifen in tamoxifen-sensitive and -resistant breast and
endometrial cancer cells.
293