UNIVERSITY OF CINCINNATI
Date:_9/4/07______
I, _Yuxin Feng______, hereby submit this work as part of the requirements for the degree of: Doctor of Philosophy in: Cell and Cancer Biology It is entitled:
Mechanism of estrogen receptor alpha action and the consequence of its conditional deletion on Mammary gland development and function
This work and its defense approved by:
Chair: Sohaib Khan, Ph.D.______Karen E.Knudsen, Ph.D.______Elwood V. Jensen, Ph.D.______Susan Waltz, Ph.D.______Nelson Horseman, Ph.D.______Robert Brackenbury, Ph.D._____
Mechanism of estrogen receptor-alpha action and the consequence of its conditional deletion on mammary gland
development and function
A dissertation submitted to the
Division of Research and Advanced Studies of the University of Cincinnati
in partial fulfillment of the requirements for the degree of
DOCTORATE OF PHILOSOPHY (Ph.D.)
in the Department of Cell and Cancer Biology
of the College of Medicine
2007
by
Yuxin Feng
M. S., Nankai University, 1997
B. S., Ocean University of Qingdao, 1990
Committee Chair: Sohaib Khan, Ph.D. ABSTRACT
ERα is a critical regulator in breast cancer and mammary gland development.
Deregulation of ER signaling correlates with abnormal mammary gland development and breast cancer. However, the role of epithelial ER remains to be clarified in vivo and the mechanism of ER signaling regulation is far from comprehensive. We hypothesize that 1) mammary epithelial ER plays critical roles in mammary gland development during pregnancy and lactation and that 2) novel, as yet identified factors in ER transcriptional regulation are involved in breast cancer development. The loxP-Cre system was used to generate epithelial ERKO mice. The well characterized MMTV-Cre and WAP-Cre transgenic mice were used to delete ER in mammary epithelial cells at different developmental stages. Early expression of MMTV-Cre arrested mammary gland
development at the neonatal stage. Successive pregnancy and lactation activated
epithelial ER ablation, which compromised side-branching, alveolar development, and
epithelial proliferation. Further analysis revealed a massive loss of luminal epithelial cells
presumably caused by apoptosis. The abnormal mammary gland development decreased
milk production, thereby, caused growth retardation in the offspring. Similar phenotypes
were also observed in MMTV-ERKO females in lactation. Thus, we concluded that
epithelial ER is essential for mammary gland development during pregnancy and
lactation stages. To further pursue the molecular mechanism of ER signaling regulation, a
human mammary gland cDNA library was screened to identify novel factors that interact
with ER. One novel ERα binding protein identified in the screen contains two conserved
LXXLL motifs (NR-box) and a coiled-coil domain. The protein product, which we
named NRCC, consists of 3 isoforms that vary in their N-terminal region. NRCC is
2 conserved in vertebrates and its mRNA was detected in human breast cancer cells and mouse breast tumors. We found that NRCC-A interacts with ERα and enhances ERα transcriptional activity in human cancer cells. Moreover, NRCC-A co-localized with ERα in the cell nucleus and was recruited to ER target gene promoters. SiRNA analysis indicated that NRCC proteins are important for endogenous ERα-mediated transcriptional activity and estrogen dependent cell proliferation. Taken together, these data indicate that
NRCC-A is a novel coactivator for ERα.
3 4
DEDICATION
To my grandparents
and
my father
5 ACKNOWLEDGMENTS
So many brilliant and giving people guided and supported me for my research. I would like to specifically thank my advisor Professor Sohaib Khan for laying the foundation for this study. I am grateful for his guidance and patience in these years. He has been a great advisor and mentor. His commitment, determination and dedication shape me to become a scientist.
I am sincerely grateful to the following persons and institutions:
To my wife, my mother, and my daughters (Selena and Hannah) for their support and patience;
To my committee for their brilliant advice and enlightened discussion: Drs. Elwood Jensen, Nelson Horseman, Robert Brackenbury, Susan Waltz and Karen Knudsen;
To the current and previous members of Khan lab for their help and friendship: Drs. David Manka, David Singleton, Gina Bell, Elizabeth Shaughnessy, Robin Tharakan, Jun Yang, Peihong Jin and Pierig Lepont;
To the Dept. of Cancer and Cell Biology and Cell and Molecular Biology graduate program:
Management team: Susan Seiler, Sasha Simms, Jaynee Tolle, Barbara Carter, Amy Itescu;
Zhang lab; Ip lab; Stambrook lab; Ben-Jonathan lab; Eric and Karen Knudsen lab;
Joe Closson: without whom our network would not run;
UC Center for Biological Microscopy: Birgit Ehmer and Nancy Kleene;
To all the Graduate students in Cancer and Cell Biology;
To the UC Gene Targeting Core: Phil Sanford and Dr. Tom Doetschman
To the UC Comparative Pathology: Rita Angel and Dr Greg. Boiwin
To the Children’s Pathology Core: Lisa and Dr. Stringer;
To the National Institutes of Health Grant T32 HD07463;
To my family;
To all my friends.
6
TABLE OF CONTENTS
Abstract…………………………………………………………………………………....2 Acknowledgments…………………………………………………………………………6 Table of contents…………………………………………………………………………..7 List of figures……………………………………………………………………………...9 Abbreviations…………………………………………………………………………….11
Chapter I: Introduction…………………………………………………………………...12 A. Breast cancer and estrogen receptor.………………………………………...13 B. Estrogen signaling……………………………………………………………15 C. Estrogen receptors……………………………………………………………16 D. Hormone receptor co-factors………………………………………………...17 1. Corepressors………………………………………………………...... 17 2. Coactivators……………………………………………………………...18 i. P160 family…………………………………………………..19 ii. Co-integrators………………………………………………..20 iii. TRAP/mediators……………………………………………..20 iv. Chromatin remodeling factors……………………………….21 v. Secondary coactivators………………………………………21 vi. Other coactivators……………………………………………22 E. Morphogenesis of mammary gland development……………………………23 F. ERα in mammary gland development……………………………………….25 G. ER-related signaling pathways in mammary gland development…………....26 H. Introductory conclusions and hypothesis…………………………………….29 I. References……………………………………………………………………32
Chapter II: NRCC-A enhances estrogen receptor alpha (ERα) transcriptional activity……………..43 A. Abstract………………………………………………………………………44 B. Introduction…………………………………………………………………..45 C. Results………………………………………………………………………..47 D. Discussion……………………………………………………………………53 E. Materials and methods……………………………………………………….57 F. References……………………………………………………………………60 G. Figures and legends…………………………………………………………..63
Chapter III: Estrogen receptor-α expression in the mammary epithelium is required for ductal and alveolar morphogenesis in pubertal, pregnant and lactating mice……………………….73 A. Abstract………………………………………………………………………74 B. Introduction…………………………………………………………………..76 C. Results………………………………………………………………………..79 D. Discussion……………………………………………………………………85
7 E. Materials and methods……………………………………………………….89 F. References……………………………………………………………………92 G. Figures and legends…………………………………………………………..95
Chapter IV: Mechanism of ERα-dependent developmental changes in mammary gland…………...109 A. Abstract……………………………………………………………………..110 B. Introduction…………………………………………………………………111 C. Results………………………………………………………………………115 D. Discussion…………………………………………………………………..119 E. Materials and methods……………………………………………………...124 F. References…………………………………………………………………..126 G. Figures and legends…………………………………………………………130
Chapter V: Summary and conclusions………………………………………………….139 A. Summary……………………………………………………………………139 B. Conclusion…………………………………………………………………..139 C. References…………………………………………………………………..150
Chapter VI: Future directions…………………………………………………………..153 A. The role of ERα in AIB1-induced tumors………………………………….153 B. The anti-inflammation effect of ERα in AIB1-induced mammary tumorigenesis……………………………………………………………….154 C. The role of ERα in mammary stem cells and epithelial progenitor cells…...156 D. References…………………………………………………………………..158
8
List of figures:
Chapter I: Introduction Figure 1 Estrogen induced cascade of cellular events Figure 2 Functional domains of ER Figure 3 Transcription cycle of estrogen-dependent activation of pS2 gene Figure 4 Stages of postnatal mouse mammary gland development Figure 5 ERα signaling in mammary gland development
Chapter II: NRCC is a novel ERα coactivator Figure 1 NRCC protein structure and mRNA expression in different tissues and cells Figure 2 NRCC-A interacts with ERα in mammalian cells Figure 3 NRCC-A enhances ERα and AR transcriptional activity Figure 4 NRCC-A and ERα co-localized in cell nucleus Figure 5 NRCC-A is a nuclear receptor coactivator Figure 6 NRCC-A promotes cancer cell proliferation
Chapter III: Estrogen receptor-α expression in the mammary epithelium is required for ductal and alveolar morphogenesis in pubertal, pregnant and lactating mice Figure 1 Targeted and conditional disruption of the mouse ERα gene using the Cre-loxP recombination system Figure 2 ERα is required for mammary gland development Figure 3 Dilation and inadequate branching of ducts in WAP-ERKO mice during 2nd lactation Figure 4 Growth retardation in WAP-ERKO pups from the 2nd litter is attributable to mother’s genotype Figure 5 Loss of ERα and PR immunohistochemical staining in WAP-ERKO (WAP fl/fl), but not ERαfl/fl (fl/fl) mammary glands of the 2nd lactation, day 1 Figure 6 Introduction of loxP sites into the ERα locus Figure 7 Abnormal lubuloalveolar development and ductal branching during first lactation cycle in WAP-ERKO mice Figure 8 Detection of whey acidic proteins in WAP-ERKO and control mice Figure 9 ER and PR expression in virgin WAP-Cre / ERαfl/fl mammary gland Figure 10 Normal ER expression in mammary stromal cells and uteri of WAP- ERKO females
Chapter IV: Mechanism of ERα-dependent developmental changes in mammary gland Figure 1 Mammary ductal structure and mammary epithelial cells Figure 2 Ductal dilation and lack of side-branching in MMTV-ERKO females during lactation Figure 3 Abnormal alveoli in MMTV-ERKO mice during lactation Figure 4 Normal CK14 and CK18 expression in WAP fl/fl virgins Figure 5 Loss of luminal epithelial cells in WAP-ERKO females in 2nd lactation
9 Figure 6 Loss of CK5 negative cells in the WAP-ERKO females in second lactation Figure 7 Apoptotic epithelial cells in WAP-ERKO mammary gland
Chapter V: Summary and Conclusions Figure 1 Ductal dilation in PRKO, WAP-ERKO and PRLKO mammary gland Figure 2 Epithelial ER and its targets are required for epithelial proliferation
10 Abbreviations
αERKO/ERαKO, estrogen receptor alpha knockout; AF, transcriptional activation domain; AIB1, amplified in breast cancer 1; AR, androgen receptor; bZIP, basic leucine zipper; CBP; CREB binding protein; C/EBPβ, CCAAT/enhancer binding protein β ; Cre,
Cre-recombinase; CK, cytokeratin; CoCo-A, coiled-coil coactivator-A; ER or ERα, estrogen receptor alpha; ERβ, estrogen receptor beta; FOXA1, forkhead box A1 ; KO, knockout; E, estrogen; ES cell, embryonic stem cell; fl/fl, ERαfl/fl; HAT, histone acetyl transferases; HDAC, histone deacetylase; IGF, insulin-like growth factor; LBD, ligand binding domain; IKKα, inhibitor of κB kinase α; LCoR, ligand-dependent corepressor;
MMTV, mouse mammary tumor virus; mTOR, mammalian target of rapamycin; NCoR, nuclear receptor corepressor; NR, nuclear receptor; NR-box, LXXLL motifs; NRCC, nuclear receptor coiled-coil coactivator; P, progesterone; PCNA, proliferating cell nuclear antigen; PI-MEC, parity-induced mammary epithelial cell; PR, progesterone receptor; PRL, prolactin; RANK, receptor activator of NF-κB; RANKL, RANK ligand;
REA, repressor of estrogen receptor activity; Rip140, nuclear receptor interacting protein
140; siRNA, small interfering RNA; SMRT, silencing mediator (corepressor) for retinoid and thyroid-hormone receptors; SRC, steroid receptor coactivator; Stat, signal transducers and activators of transcription; TEB, terminal end bud; TGF-β, transforming growth factor β; TRAP, thyroid receptor associated protein; WAP, whey acidic protein.
11
Chapter I
Introduction
12 A. Breast cancer and estrogen receptor
Breast cancer is a devastating disease and the second leading cause of cancer death
among women, accounting for more than 350,000 deaths per year worldwide (Holst et al.,
2007). Prompted by the observation that breast cancer growth varied with estrous cycle,
Sir George Beatson performed oophorectomy in breast cancer patients and observed
favorable response (Beatson, 1896). This was the first hint that female sex hormones are
involved in breast cancer growth. Estrogens, first isolated by Edward A. Doisy and his
colleagues (MacCorguodale et al., 1936), play an important role in the normal physiology
of the breast and have a well-established proliferative effect upon breast tissue (Levenson
et al., 2001; Struse et al., 2000). Growth and progression of many breast cancers are
dependent upon estrogen (Leygue et al., 2000; Mortimer et al., 2001). The discovery of
ER by Elwood Jensen laid the foundation for anti-estrogen treatment (Jensen and
Jacobson, 1962). Furthermore, the application of ER as a prognostic marker in breast
cancer originated by Dr. Jensen benefited breast cancer patients in selecting their type of
treatment (Jensen et al., 1971). Breast cancer is a heterogeneous disease in which the
stable phenotypes develop during tumor progression in each subtype (Polyak, 2006).
Depending on its origin, breast cancer can be divided into stromal derived and epithelial
derived breast cancers. The majority of breast cancers are derived from epithelial cells. In
epithelial derived breast cancers, most originate from luminal epithelial cells and the ones
derived from myoepithelial (basal) cells are uncommon (Barsky and Karlin, 2005; Polyak and Hu, 2005). The most important determinants of breast cancer subtypes are the
presence or absence of ER and progesterone receptor (PR), the amplification and
overexpression of the HER2 oncogene, and histological grade. More recently it was also
13 reported that ERα gene is frequently amplified in breast cancer (Holst et al. 2007). Based
on these features, breast tumors are divided into luminal A, B, and C, HER2+, and basal
subtypes (Finnegan and Carey, 2007; Sorlie et al., 2003). Luminal derived breast cancers
are ER-positive, while HER2+ and basal derived are generally ER negative. Luminal A
subtype has the highest expression of ER and ER regulated gene expression and a better
clinical outcome compared to B group which has the worst clinical outcome (Sorlie et al.,
2003; Sotiriou et al., 2003).
The biological actions of estrogen are mediated by the products of two genes within the
nuclear receptor family, ERα and ERβ (Saji et al., 2000). The presence of ER correlates positively with the response of patients to therapy with antiestrogens such as tamoxifen
(Dickson and Stancel, 2000). The absence of ER is generally associated with faster growing and more aggressive tumors that are non-responsive to tamoxifen therapy (Mann et al., 2001). Although tamoxifen is considered an effective mode of therapy in ER- positive breast cancers, it has been observed that a significantly high percentage of patients develop resistance to this agent within five years. Only about 30% of the total patients treated with tamoxifen show complete or partial remission that lasts for a year or more (Muss, 1992). Among the ER-positive tumors that initially respond to tamoxifen therapy, a majority develop resistance to antiestrogen without change in ER expression.
Although not entirely clear, upregulation of signaling growth factors (e.g. IGF-1, HER2) and ER coactivators (AIB1, cyclin D1), has been implicated as possible explanation
(Osborne et al., 2003; Osborne et al., 2005; Parisot et al., 1999; Wang et al., 2006). In some breast tumors, ER signaling might be deregulated as reflected by the loss of PR, a
14 known ER-target gene. Overexpression of ER coactivator, AIB1 might be related to loss
of PR (Bardou et al., 2003; Torres-Arzayus et al., 2004). In addition, about 33% of ER-
positive tumors fail to respond to tamoxifen and are considered to be estrogen- independent and tamoxifen resistant (Connor et al., 2001). It is possible that the varied results obtained with antiestrogen therapy are contributed by the differences in ER
function. These observations emphasize the importance of ER in breast cancer and
warrant further investigation towards understanding the mechanism of ER action in
mammary gland physiology and disease.
Many crucial factors in breast cancer are also vital for mammary development (Wiseman
and Werb, 2002). In a workshop in 2003, National Cancer Institute emphasized that “The
study of mammary carcinogenesis can’t move forward unless the fundamentals of normal
mammary development are better understood.” Under this guideline, we believe that it is
impossible to dissect the in vivo role of ER in breast cancer development without understanding the role of ER in mammary gland development. Thereby we explore the role of ER in adult mammary gland development employing loxP-Cre conditional knockout system.
B. Estrogen signaling
Estrogen is a steroid hormone that functions as the principal mediator of reproductive function in female mammals. Its role in stimulation of growth and proliferation of the
reproductive organs: uterus, vagina, and breast are well established. Estrogens bind to ER,
which acts as a transcription factor to alter the expression of estrogen responsive genes.
The central tenets of this pathway involve ligand binding to ER, receptor dimerization,
and subsequent binding to specific regulatory regions of DNA. Once bound to DNA,
15 additional proteins, known as coactivators, are recruited to the site. These coactivators are
believed to interact with basal transcriptional machinery to turn on the expression of the
target genes (Pike et al., 2000), whose Fig. 1 Estrogen-induced cascade of cellular events products are necessary for cell growth
and differentiation (Nephew et al.,
1993; Webb et al., 1993) (Fig. 1). In
this manner, hormone binding to ER
acts like a switch to trigger the cascade
of events, which culminates in the
growth and differentiation of a wide variety of tissues in the body for specialized
functions.
C. Estrogen receptors
Estrogen receptors belong to the nuclear receptor (NR) superfamily of ligand-activated transcription factors. Of the two ERs, ERα and ERβ, the former is considered the main
receptor for mammary gland development and function. ERα, a protein comprising of
595 amino acids, can be divided into six regions denoted A-F (Fig 2). The A/B region possesses one of the two activation domains, AF-1. Region C is the DNA binding domain and contains two zinc fingers. Region D contains the nuclear translocation signal.
Region E of the ERα is the hormone-binding domain, which is responsible for ligand
binding, receptor dimerization, transcriptional activation (AF-2) and interaction with coactivators (Klinge, 2000; Nilsson et al., 2001). The F-domain of the ERα may have a specific modulatory function that affects the agonist/antagonist effectiveness of antiestrogens (Kim et al., 2003b). The gene encoding ERα is large (~140 kb) and
16 organized into eight axons. Exon 1 encodes most of the A/B region; Exon 2 and 3 encode
region C; exon 4 encodes part of region C, all of region D and part of region E; the rest of
the E domain and the
entire F-domain is
encoded by exon 8
(Green et al., 1986).
Ligand-induced changes
in the conformation of
the E-domain seem to
initiate the recruitment of Fig. 2 Functional Domains of ER (Klinge C 2000) coactivators for ER
function (McDonnell and Norris, 2002; Nilsson et al., 2001).
D. Hormone receptor co-factors
The full activity of NRs depends on a large number of factors that do not bind to DNA
directly, but are recruited to promoters by the NRs (Dennis and O'Malley, 2005;
Rosenfeld et al., 2006). These proteins are collectively defined as co-factors or coregulators. The co-factors are broadly divided into corepressors and coactivators.
1. Corepressors
Some co-regulators are silencing mediators that either contain intrinsic transcription silencing activity or can bridge the interaction of NRs with other silencing factors. There are two types of corepressors, ligand-dependent and ligand-independent nuclear receptor corepressors. In the absence of ligand, NRs interact with silencing mediators, such as
NCoR/SMRT which can recruit corepressor complex including histone deacetylases
17 (Fernandes et al.), GPS-2 and TBL-1 to the promoters of NR target genes and shut down
target gene expression (Perissi et al., 2004; Zhang et al., 2002). The interaction of
LBD/AF2 domain of NR with NCoR and SMRT is mediated by a conserved motif,
LXXI/HIXXXI/L (Hu and Lazar, 1999; Webb et al., 2000). Ligand-dependent
corepressors are recruited by ER through LXXLL motifs (NR-boxes) in the presence of ligand. MTA-1s, REA, RIP140 and LCoR are each recruited to ER in a ligand-dependent
manner (Delage-Mourroux et al., 2000; Fernandes et al., 2003; Kumar et al., 2002; Mussi
et al., 2006; Park et al., 2005). MTA-1s inhibits ER transcriptional activity by
sequestering ERα in the cytoplasm (Kumar et al., 2002). LCoR represses NR
transcription in HDAC dependent and independent mechanisms (Fernandes et al., 2003).
Among the ligand-dependent corepressors, REA is the only one that was characterized in
vivo. It inhibits ER target gene expression in mammary gland and uterus. Importantly, it
modulates ER activity in mammary gland development and might be related to breast
cancer development (Mussi et al., 2006; Park et al., 2005).
2. Coactivators
NR coactivators also interact with hormone receptors in ligand-dependent and ligand-
independent manners. Cyclin D1 and CIA bind to ER in the absence of ligand and
enhance ERα transcriptional activity (Sauve et al., 2001; Zwijsen et al., 1998; Zwijsen et
al., 1997). However, most classical NR coactivators bind to NR in the presence of ligands.
Using the ligand-binding domain of ER as bait, O’Malley and colleagues identified the
ER-interacting proteins as steroid receptor coactivators (SRCs) (Kamei et al., 1996;
Onate et al., 1995). Since then, approximately seven classes of coactivator complexes
have been identified that interact with liganded-ERα: (i) p160 family, SRC-1/NCoA-1,
18 SRC-2/NCoA-2/TIF2/GRIP1, and SRC-3/AIB1/NCoA-2/ACTR/RAC3/pCIP/TRAM-1;
(ii) Co-integrators (histone acetyl transferases (HATs) such as CBP/p300); (iii)
TRAP/mediators; (iv) Chromatin remodeling factors eg. SWI/SNF; (v) Secondary
coactivators; (vi) Histone arginine methyl transferases (HMTs), eg. CARM1. (vii) Other
coactivators. In ligand-dependent interactions, ligand binding promotes a conformation
change in ER which creates a contact surface for LXXLL motifs on co-factors. Many
coactivators directly bind to NR ligand binding domain through NR-boxes (McDonnell and Norris, 2002; Rosenfeld et al., 2006). Conversely, upon antiestrogen binding, helix
12 of ER is positioned such that corepressor recruitment is favored, which antagonizes
ER activity (Jepsen and Rosenfeld, 2002). However, the interaction of co-regulators with
ER is dynamic and well orchestrated (Perissi et al., 2004; Rosenfeld et al., 2006). The remarkable progress made towards understanding the intricate mechanism of ER action has also paved the way to exploit this knowledge to better understand disease processes associated with the receptor and/or its coactivators. The major groups of coactivators are summarized in the following paragraphs.
i. P160 family
SRC1 (steroid receptor coactivator 1), A member of P160 family, was the first identified
NR coactivators (Kamei et al., 1996; Onate et al., 1995; Xu and Li, 2003). The three members in p160 family of proteins, SRC1, SRC2 (TIF-2) and SRC3 (AIB1) share high amino-acid-sequence identity and have conserved functional domains，containing an N-
terminal bHLH/PAS domain that interact with secondary coactivators (Chen et al., 2005;
Kim et al., 2003a), multiple NR-boxes in the central region of the protein, C-terminal
AD1 domain binding to p300/CBP and histone acetyltransferase (HAT) (Xu and Li,
19 2003). Upon ligand binding to NRs, p160 members are the first coactivators recruited by
NRs and function as platform for the docking of other coactivators (CARM1, p300/CBP and secondary coactivators) to the ligand-bound NRs (Li et al., 2000; Voegel et al., 1998).
Gene disruption experiments revealed important biological functions of SRC family members in reproduction system, metabolism, energy balance and hormonal signaling
(Gehin et al., 2002; Picard et al., 2002; Wang et al., 2000; Xu et al., 2000; Xu et al.,
1998).
ii. Co-integrators
CREB binding protein (CBP) and its homologous protein p300 are large evolutionary conserved proteins that serve as coactivators for different types of transcription factors
(Xu, 2005). Recruited to ER by p160 coactivators and directly interacting with ER through its N-terminal NR-box, CBP and p300 are acetyltransferases that acetylate histones as well as SRCs (Chen et al., 1997; Spencer et al., 1997). The acetylation of
SRC coactivators by CBP facilitates the abrogation of SRC coactivators from ligand- bound NRs. Notably, the null mice of CBP and P300 are embryonic lethal, confirming they are broadly acting coactivators (Tanaka et al., 2000; Yao et al., 1998).
iii. TRAP/mediators
TRAP/mediators is a large multisubunit protein complex that bridges the direct communication between nuclear receptors and the general transcription machinery
(Reviewed in Malik and Roeder 2000, Rosenfeld et al. 2006). Like many other coactivators, the NR interaction unit in the complex, TRAP220 (thyroid receptor- associated protein) interacts with NRs through the NR-box and is essential for ER transcriptional activity and ER-positive breast cancer proliferation (Ito et al., 2000; Zhang
20 et al., 2005). The sequential recruitment of p160 coactivator and CBP may facilitate the
interaction of Trap220 with NRs, coordinating the assembly of NR and mediator complex
(Metivier et al. 2003, Shang et al. 2000). Unlike SRCs, Trap220 null mice are embryonic
lethal, indicating the role of this protein is beyond NR coactivators (Ito et al., 2000).
iv. Chromatin remodeling factors
The ATP-dependent chromatin-remodeling complexes use energy from ATP hydrolysis
to increase the mobility of nucleosomal DNA, thereby either restricting or permitting
binding of transcription factors and subsequent assembly of functional pre-initiation
complexes (Becker and Horz, 2002). The mammalian homolog of SWI/SNF chromatin
remodeling complex associates with NR in the coregulator exchange cycle (Becker and
Horz, 2002; Garcia-Pedrero et al., 2006; Link et al., 2005). Presumably, they interact with
ER during the stage of exchange of coactivators and the coactivator clearance stage
(Rosenfeld et al., 2006). BAF57, a subunit in the chromatin remodeling complex
mediates interactions with ER and AR (Belandia et al., 2002; Garcia-Pedrero et al., 2006;
Link et al., 2005). BAF57 also interacts with SRC family members and is necessary for
their ability to stimulate transcription by ER (Belandia et al., 2002).
v. Secondary coactivators
Some coactivators do not interact with NR directly, but enhance NR transcriptional
activity through binding to bHLH/PAS domain of p160 family members (Chen et al.,
2005; Kim et al., 2003a). CoCoA, a coiled-coil domain secondary coactivator can not
enhance NR transcriptional activity by itself, but enhances NR transcriptional activity in
a SRC1- dependent manner (Chen et al., 2005; Kim et al., 2003a).
21 vi. Other coactivators
There are a large number of coactivators that do not belong to the known groups of coactivators, such as SRA, Ubc9, E6AP and ERAP140 (Gottlicher et al., 1996; Lanz et al., 1999; Nawaz et al., 1999; Shao et al., 2002). Some of these have unique biological functions other than enhancing ER transcriptional activity. For example, E6AP is involved in proteasome-dependent protein degradation (Nawaz et al., 1999). However, the biological functions of many coactivators in this growing group remain to be defined.
BAF BRG1 p160 HMT p300 PRMT5 HDAC
BAF Activated promoter N-CoR M A complex BRG1 M M PCAF H3 H4
N-CoR Silent promoter complex M HMT H3 H4
26S Proteosome p160 HAT
Elongation p160 HAT complex M A M A Mediator M A Pol II H3 H4
TAFs
A A PARP1 M A CBP H3 H4
Figure 3. Transcription cycle of estrogen-dependent activation of pS2 gene. Some of the key co-regulators were shown in a simplified ER transcription activation process. In the absence of estrogen, estrogen receptor binds to N-CoR and repressor complex. Upon binding to estrogen, p160 family coactivator was recruited to replace N-CoR repressor complex, recruiting p300 and other histone remodeling enzymes to the coactivator complex and trigger the modification of histones. Subsequently, the Trap220 mediators bridge the RNA Pol II and basic transcription factors with ER and start the transcription. Ligand bound ER was then released from the pS2 promoter for degradation. 26S proteosome mediated protein degradation and ubiquitination are required for the cycling of co-factors and ER.
22
E. Morphogenesis of mammary gland development
Distinct from other organs, the structure of mammalian breast tissue continually changes throughout the female reproductive cycles. The process of postnatal mammary gland development has been described in several excellent reviews (Hennighausen and
Robinson, 1998, 2005; Hovey and Trott, 2004). In brief, the mouse mammary epithelia form a small ductal network (mammary primordia/placode) before birth, which remains dormant until puberty (Hennighausen and Robinson, 1998). In response to the changing hormonal milieu at puberty, the rudimentary ductal network in the mammary gland undergoes massive arborization and invades the fat pad. Mammary gland development is not complete until pregnancy and lactation occur: alveolar structures form on the ductal tree and differentiate into lobular alveoli which will produce milk for pups during lactation. At weaning, the secretory epithelia of the mammary gland undergo apoptosis, the fat cells redifferentiate, and the gland is remodeled back to a state morphologically resembling that of the adult virgin (Hennighausen and Robinson, 1998; Ismail et al., 2003;
Richert et al., 2000) (Figure 4). The post-pubertal mammary gland development is under tight control of hormones (e.g., estrogen, progesterone, prolactin, growth hormone) and growth factors (e.g., IGF-1, FGF and EGF etc) (Fendrick et al., 1998; Lyons et al., 1958;
Topper and Freeman, 1980). Ovarian hormones, including estrogen and progesterone, control ductal outgrowth and alveolar proliferation (Hewitt et al., 2005; Ismail et al.,
2003; Vomachka et al., 2000). ERα and another hormone nuclear receptor, Progesterone receptor (PR) are known to promote mammary epithelial cell and breast cancer cell proliferation (Ismail et al., 2003; Nilsson et al., 2001). E2 induces PR expression via ERα, suggesting the role of ERα in alveolar development is mediated by PR (Ismail et al., 2003;
23 Lamote et al., 2004). In addition to PR, prolactin (PRL) and prolactin receptor (PRLR) are also master regulators for secondary branching and lobular alveoli (Lyons et al., 1958;
Topper and Freeman, 1980; Vomachka et al., 2000)
PRKO PRLKO PRLRKO αERKO
Adult Pregnancy virgin
Neonatal Immature virgin
Involution lactation Stages of mouse mammary gland development
Figure 4 Stages of postnatal mouse mammary gland development. Distinct from other mammalian organs, the structure of mammary gland changes continually in the reproduction-active females. The mammary epithelial network fills the fat pad after puberty. Lobuloalveoli are formed during pregnancy to produce milk for the pups upon lactation. In involution, the secretory epithelia die by apoptosis and the mammary gland is remodeled to a state similar to the adult (nulliparous) females. ER and its target PR had critical roles in mammary gland development. ERα is pivotal in ductal elongation in puberty. Therefore, mammary gland development of ER knockout mice arrests in neonatal stage. PR, PRLR, and PRL are master regulators for tertiary branching and alveolar development. Mammary gland can not develop beyond adult virgin stage in the absence of PRL, PRLR or PR. (Some pictures were from http://mammary.nih.gov/ by Lothar Hennighausen)
development during pregnancy and lactation stages (Goffin et al., 2002; Hennighausen and Robinson, 2005; Horseman et al., 1997; Hovey et al., 2002). As in PRKO mice, side- branching and alveolar development were compromised in PRL-null and PRLR- heterozygous mice (Brisken et al., 1999; Horseman et al., 1997). ER, PR and PRLR are
24 co-expressed in same epithelial cells and the expression of PRLR might also be regulated
by ER (Dong et al., 2006; Grimm and Rosen, 2003; Hovey et al., 2002; Ormandy et al.,
1992). These elegant studies, exploiting genetic knockout mouse models, reinforce the
concept that mammary gland development and function are under the tight control of
overarching hormonal signaling.
F. ERα in mammary gland development
Estrogen triggers mammary ductal elongation during puberty and stimulates alveologenesis during pregnancy (Daniel and Silberstein 1987). The findings that germ line deletion of ERα rather than ERβ disrupts postnatal mammary gland development,
established the dominant role of ERα in mammary gland development (Hewitt et al.,
2005). The rudimentary ductal network in αERKO mice failed to invade into the
mammary fat pad at puberty. Since ERα governs mammary gland development at
different levels, the interpretation of the observed phenotype was not straightforward.
First, as a consequence of germ line deletion, the pituitary gland would be devoid of ERα,
thus deregulating the expression of pituitary hormones, which have profound impact on
mammary gland development. Second, ERα is expressed in different cellular
compartments of the mammary gland. Consequently, the impairment of mammary gland development in ERKO mice may be due to the inability of the mammary gland itself to respond to the stimulation of estrogen in the absence of mammary ERα or it may be secondary to endocrine abnormalities (Bocchinfuso et al., 2000; Mueller et al., 2002).
Among the different cell-types that constitute mammary gland, the epithelial and the stromal cells occupy the central stage. In the mouse mammary gland, both cell-types express ERα (Haslam et al. 1992). The cross-talk between the mammary stroma and
25 epithelium orchestrates mammary gland development (Wiseman & Werb 2002). In order
to understand the complex paracrine/autocrine estrogen signaling in the mammary gland,
Cunha and colleagues conducted tissue recombination assays, using stromal and
epithelial tissues from WT- and ERKO mice, and concluded that the epithelial ER is
dispensable for mammary gland development/function (Cunha et al., 1997). There was
some skepticism regarding this conclusion, which was later thought to be due to the use
of tissue from a hypomorphic ERKO mice generated by Korach and colleagues (Couse et
al., 1995; Kos et al., 2002). More recently, Chambon and colleagues generated another
ER knockout mouse line and the tissue recombination studies from these ERKO mice established that epithelial, rather than stromal ERα is essential for mammary gland
development (Mallepell et al., 2006). To clarify the controversial results from the
previous studies, we have reexamined this issue in a mouse model by employing tissue-
specific ablation of ERα in the mammary epithelial compartment and providing support
for the conclusions reached by Mallepell et al.
G. ER related signaling pathways in mammary gland development
Many factors implicated in breast cancer are also vital for mammary development
(Wiseman and Werb, 2002). Hormonal and growth factor pathways are critical for postnatal mammary gland development (Hennighausen and Robinson, 1998, 2005; Oakes et al., 2006; Wiseman and Werb, 2002). Among these signaling pathways, estrogen-ERα
signaling has an irreplaceable niche in postnatal mammary gland development
(Bocchinfuso and Korach 1997, Bocchinfuso et al. 2000 and Mallepell et al 2006). Until
recently, it was thought that ER is required for ductal elongation but is not needed for
mammary gland development in pregnancy and lactation, based on the conclusions drawn
26 from the studies utilizing a hypomorphic ERKO mice (Brisken, 2002; Hennighausen and
Robinson, 2005). However, E2- and P-signaling are required for alveolar development and PR, a master regulator of alveolar development, is a direct target of ER, suggesting
ER signaling is indispensable for the mammary gland differentiation cycles (Haslam,
1988a, b; Hovey et al., 2002; Soyal et al., 2002).
Transcriptional regulation of ER in mammary gland development is not well understood.
Gene targeting experiments revealed that Gata-3 and C/EBPβ regulate ER expression in
luminal epithelial cells (Asselin-Labat et al., 2007; Grimm and Rosen, 2003; Kouros-
Mehr et al., 2006). The expression of Gata-3, a zinc finger transcription factor, is highly
correlated with ER in breast cancer cells as well as in mammary luminal epithelial cells
(Eeckhoute J, 2007; Lacroix and Leclercq, 2004; Perou et al., 2000). Required for
mammary placode formation, Gata-3 has an essential role in embryonic and postnatal
mammary gland development (Asselin-Labat et al., 2007; Kouros-Mehr et al., 2006).
There is a significant loss of ER-positive epithelial cells in Gata-3 null mammary gland,
whereas, Gata-3 binds to ER gene and enhances ER expression in breast cancer cells,
suggesting Gata-3 is critical for the upstream signaling of ERα (Asselin-Labat et al., 2007;
Eeckhoute J, 2007; Kouros-Mehr et al., 2006). Furthermore, Gata-3 regulates the
expression of FOXA1, a critical transcription activator for many ERα target gene
expression (Carroll et al. 2005; Laganiere et al. 2005). The bZIP transcription factor
family member, C/EBPβ, attenuates ER expression in luminal epithelial cells.
Consequently, knocking out C/EBPβ results in the release of this repression, allowing
uniform ER expression in luminal epithelial cells (Grimm and Rosen, 2003). These
27 observations emphasize the importance of upstream regulation of ER in the mammary
gland development and function.
Fig. 5 ERα signaling in mammary gland development
C/EBPβ Gata-3 Prl AIB1
PrlR ERα TGF-β
PR Elf-5 REA
CyclinD1 Stat5
Figure 5 ERα signaling in mammary gland development. ER is upstream signal of CyclinD1, PRLR and PR in alveolar development. TGF-β, ERα, PRLR and PR are co- expressed in normal mammary epithelial cells. TGF-β restricts the ER/PR/PRLR positive cells from proliferation. C/EBPβ inhibits ER expression, thereby, C/EBPβ positive cells are ER/PR negative. PRL and PR pathways regulate same downstream targets, such as Stat5 and RANKL, in alveolar development. The co-regulators, AIB1 and REA, regulate ER transcriptional activity in mammary gland development.
Expressing in mammary epithelial cells, ERβ and α share many target genes in the mammary gland (Heldring et al., 2007). However, ERβ inhibits ERα transcriptional activity, suggesting the inhibitory role of ERβ in breast cancer progression (Matthews and Gustafsson, 2003; Matthews et al., 2006; Speirs and Walker, 2007). Mammary gland development in ERβKO mice is relative normal, supporting the dominant role of ERα in breast cancer and mammary gland development (Forster et al., 2002).
It has been known that co-regulators regulate ER transcriptional activity in vitro since the
identification of the first coactivator SRC1 (Xu and Li, 2003). The in vivo roles of ER co- regulators have not been directly addressed until recently. Several elegant in vivo studies
28 revealed that both coactivators and corepressors of ER are critical for mammary gland
development. AIB1 is a p160 family coactivator amplified in a subset of breast cancers
and the mRNA expression is increased in up to 60% of breast cancers (Anzick et al.,
1997). Overexpression of AIB1 in transgenic mice leads to mammary hypertrophy,
hyperplasia and delayed mammary involution. Significantly, the incidence of malignant
mammary tumors and tumors in other organs is increased. Thereby, AIB1 is established
as an oncogene. AIB1 overexpression increases expression of ER and its downstream
target IGF-1, thus enhancing activity of IGF-1 downstream AKT/mTOR (Torres-Arzayus
et al., 2004). IGF-1 receptor signals synergize with ER action in human breast cancer
cells (Dupont et al., 2000a), thereby, IGF-1 signaling may also enhance ER activity in
vivo. Repressor of ER activity (REA) is a ligand-dependent corepressor of ER which co-
expressed with ER in mammary epithelial cells (Delage-Mourroux et al., 2000; Montano
et al., 1999). REA knockout mice have accelerated mammary gland development and
delayed involution. In vivo ERE-luciferase assay showed ER overactivation in mammary
gland and the expression of ER targets, PR and Cyclin D1 are increased. So far, AIB1 is the only oncogene among the identified coactivators (Torres-Arzayus et al., 2004).
Therefore, the identification of novel coactivators is critical for understanding ER
signaling regulation in mammary gland development and mechanism of breast cancer
induction.
H. Introductory conclusions and hypothesis
Taken together, our knowledge about ERα transcriptional regulation has expanded
significantly with over 200 identified NR co-regulators. Although much has been learned
about ER transcriptional control, many questions remain. How and in which
29 circumstance the ligand-dependent repressors are involved in NR activation/inactivation
in vivo is not known. The mechanism to overcome the ligand-dependent repression also begs an answer. Though ER can be activated in the absence of ligand stimulation
(Rosenfeld et al. 2006), the factors that govern ligand-independent activation are not well understood.
How coactivators are involved in breast cancer development is largely unknown. Given
the implication that ER has fundamental roles in breast cancer initiation and progression
and SRC3 (AIB1) is the only established oncogene among the NR coactivators (Torres-
Arzayus et al. 2004), we predict that some novel coactivators might also be involved in
breast cancer development. We hypothesize that novel, as yet identified factors in ER
transcriptional regulation are involved in breast cancer development. To address this
hypothesis, we and our collaborators conducted a yeast two-hybrid screen and were able
to identify some potential novel co-regulators of ERα.
Since deregulation of normal mammary gland development likely leads to breast cancer,
characterizing the role of ER in this process will provide an essential contribution
towards understanding breast oncogenesis. Moreover, this approach could aid in
development of novel therapeutic strategies. Although mammary reconstitution
experiments have indicated that epithelial ER is required for normal gland development,
critical questions remain to be answered, including: 1) Whether ER is required for breast
cancer initiation and progression. 2) If epithelial ER is critical for mammary gland
development during pregnancy, lactation and involution and 3) Whether AIB1 induced
breast cancers are ER dependent.
30 Our tissue specific ER conditional knockout animal model will allow these questions to be addressed. Based on the current knowledge of ER’s in vivo activity, we developed the hypothesis that mammary epithelial ER exerts crucial functions in mammary gland development during pregnancy and lactation. We tested this hypothesis by developing and analyzing our ER conditional animal models.
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42
Chapter II
NRCC enhances estrogen receptor alpha (ERα)
transcriptional activity
43
Abstract
Estrogen receptor alpha (ERα) is an important diagnostic marker and therapeutic target in breast cancer. Abnormal ERα activation and expression are associated with a majority of breast cancers. A key coactivator of ERα in the mammary gland, AIB1, is characterized as an oncogene. The mechanism(s) in which accessory proteins such as AIB1 affect breast cancer development are not well understood. We have characterized a novel ERα-binding protein identified in a yeast two-hybrid screen, which contains two conserved LXXLL motifs (NR-box) and a coiled-coil domain. The protein product, which we named nuclear receptor coiled-coil coactivator (NRCC), consists of 3 isoforms that vary in their N-terminal region. Conserved in vertebrates, NRCC mRNA was detected in human breast cancer cells and mouse breast tumors.
We found that the longest isoform of NRCC, NRCC-A, interacts with ERα and enhances ERα transcriptional activity in human cancer cell lines. Moreover, NRCC-A co-localized with ERα in the cell nucleus and was recruited to ER target gene promoters. Small interfering (siRNA) analysis indicated that NRCC proteins are important for endogenous ERα-mediated transcriptional activity and estrogen-dependent cell proliferation. Taken together, these data indicate that NRCC-A is a novel coactivator for ERα.
44
Introduction
Estrogen signaling is mediated by two nuclear receptors, ERα and ERβ, which regulate a
broad range of biological processes (Reviewed in (Couse and Korach, 1999; Deroo and
Korach, 2006; Koehler et al., 2005)). Like other nuclear hormone receptors, ERα contains
an N-terminal transactivation domain (AF1), a DNA binding domain, a hinge region and
a ligand binding domain overlapping with the second transactivation domain (AF2). In
the classical genomic pathway, upon binding to estradiol, dimerized ERα recruits
coactivators and a transcriptional activation complex to estrogen receptor responsive
elements (EREs), which initiates transcription of target genes. The overall effect in
responsive tissues, including breast cancer cells is to promote growth and proliferation
(McDonnell and Norris, 2002).
Nuclear receptor coactivators contribute to ERα dependent gene expression by
acetylating and methylating histones, remodeling chromatin structure, recruiting and
modifying other coactivators, and bridging the general transcription machinery with the
nuclear receptors (reviewed in Rosenfeld et al., 2006; McDonnell and Norris 2002).
Ligand binding promotes a conformation change in ER, which creates a contact surface for LXXLL motifs (NR-boxes) on co-factors (Heery et al., 1997; Voegel et al., 1998).
Some coactivators enhance ER transcriptional activity in the absence of ligand when recruited to the AF1 or LBD/AF2 domains of ERα (Sauve et al., 2001; Zwijsen et al.,
1998; Zwijsen et al., 1997).
45 Six major groups of nuclear receptor coactivators have been characterized, which
contribute to ER transcriptional activity. These include: 1) P160 family coactivators
(SRC1, TIF-2 and SRC3), which have intrinsic histone acetyl-transferase (HAT) activity
and recruit CBP/p300 to the ER transcriptional complex (Xu and Li, 2003); 2) The
integrators, CBP/p300 family members that acetylate p160 coactivators and histones and
are responsible for the quick dispersal of the coactivator complex (Kamei et al., 1996;
Yao et al., 1998); 3) DRIP/TRAP mediators, which link the general transcription
machinery with nuclear receptors (Zhang et al., 2005); 4) Chromatin remodeling proteins
(Brm and BRG-1) (Xu, 2005); 5) The non-p160 family members (Rip140, TIF1 and
ARA70), including some co-factors that can be coactivators or corepressors in different
promoter or nuclear receptor contexts (McDonnell and Norris, 2002); and 6) The
secondary coactivators, which bind to p160 family members but do not directly interact
with NRs (CoCoA and GAC63) (Chen et al., 2005; Kim et al., 2003a).
Although coactivators have an indispensable role in ERα signaling, AIB1 is the only
identified oncogene among the known coactivators (Torres-Arzayus et al. 2004). Some
ER coactivators are also important in other signaling pathways, indicating that they
mediate cross-talk between signaling networks (Rosenfeld et al., 2006). To test our
hypothesis that un-identified coactivators may have important roles in breast cancer
development and mediate cross-talk between ERα and other signaling pathways, we
conducted a screen for novel co-factors, utilizing the CDEF domains of ERα as bait. Here,
we characterize one of the identified novel binding partners of ERα, which enhances
receptor-mediated transcriptional activity and also promotes ERα dependent and
independent cancer cell proliferation.
46 Results:
Yeast two-hybrid screen identifies NRCC protein as an ERα-interacting partner:
To identify novel co-factors of ERα, a yeast two-hybrid screen was performed in a human mammary gland cDNA library with ERα CDEF domain as the bait. A number of known and novel proteins were identified in the screening, including the p160 family member
SRC1, and transcription factor Stat3. One of the unknown proteins identified in the screen is coded by a gene on chromosome 10q25.1 (gene designation C10ORF78, accession no. NM_001002759). Relatively little is known about this protein. However, based on the presence of a predicted coiled-coil domain and two NR-boxes, we named it nuclear receptor coiled-coil coactivator (NRCC). The three isoforms submitted to
GenBank were named NRCC-A, B, and C based on the length of the predicted proteins, with the 307aa NRCC-A being the longest (figure 1 A).The deduced NRCC-A protein is conserved in vertebrates, including mammals, birds, zebra fish and Xenopus (Figure 1B and supplementary figures). From amino acid 67-307, about 74% of amino acids are identical among human, chimpanzee, monkey and dog. About 76% of amino acids are conserved among all 5 mammalian species (Figure 1B). Notably, both LXXLL motifs
(NR-boxes) are conserved among the mammals. The 100% identical region among different species including the first NR-box (231-235aa) and its flanking sequence indicates the functional importance of this domain. The sequence of the second NR-box and flanking region is identical in 4 species, excluding mouse. Coiled-coil domains mediate protein-protein or protein DNA interaction (Burkhard et al., 2001; Gruber and
Lupas, 2003). CoCo-A, a secondary coactivator containing a coiled-coil domain, was found to enhance ER and AHR transcriptional regulatory activity (Kim et al., 2003).
47 Similar to the classical hormone receptor coactivators, NRCC contains two conserved
NR-boxes (LXXLL) in the C terminal region (Figure 1 A and B), which might mediate
the interaction with hormone receptors (Heery et al., 1997). Although a nuclear
localization signal was not identified, several sub-cellular localization prediction
programs (WoLFPSORT, LOCtree and SignalP3.0) predicted NRCCs as non-secretory
proteins localized in cell nucleus, containing no DNA binding domain. Taken together,
the domains within NRCC are indicative of hormone-receptor binding and a nuclear function, consistent with coactivator activity.
NRCC mRNA is expressed in human breast cancer cells.
The information from GenBank indicated that human NRCC mRNA was detected in
multiple sex hormone responsive tissues including mammary gland, male and female
reproductive systems and prostate. Mouse NRCC mRNA was detected in the mammary
tumor of MMTV-int1 transgenic mice, suggesting possible involvement of NRCC in
breast cancer development. To address the role of NRCC in ER positive breast cancer
cells, we examined the expression of NRCC in several human cancer cell lines. NRCC
mRNA was detected in ERα positive MCF7 breast cancer cells as well as Ishikawa
endometrial adenocarcinoma cells (Figure 1 C). The NRCC transcripts were also detected
in human skeletal muscle and fetal brain. The overlapped expression of NRCC with ERα
suggests possible functional interaction between the two proteins.
NRCC proteins interact with ERα in mammalian cells.
48 To confirm the interaction of NRCC with ERα in mammalian cells, the cDNAs of NRCC
were cloned into p3XFLAG-CMV-7.1 mammalian expression vector and transfected into
Ishikawa human endometrial cancer cells. As predicted, a 37kD band was detected with
Anti-Flag M2 antibody (Sigma), indicating the fusion protein was expressed in mammalian cells (Figure 2A). The interaction of ERα and NRCC proteins was then validated with Co-IP in Ishikawa cells. Both NRCC-A and NRCC-C interacted with ERα in the presence and absence of E2 (Figure 2B and data not shown). Immunoprecipitation with both anti-Flag and anti-ERα antibody showed that ERα and NRCC can pull each other down, indicating direct or indirect interaction possibly through the conserved NR- boxes of NRCC. To determine whether NRCCs can bind to other nuclear receptors, Co-
IP experiments were performed with protein extracts from Ishikawa cells co-transfected with androgen receptor (AR) and NRCC-A. Similar results to ER and NRCC-A Co-IP were obtained. AR associated with NRCC-A in both the presence and absence of dihydrotestosterone (DHT) (data not shown).
We further confirmed the interaction between ERα and NRCCs with mammalian two- hybrid assay. The NRCC cDNAs were subcloned into pM vector as bait. The SRC-1 cDNA fragment containing NR-boxes known to interact with ERα was used as positive
control. In the absence of ERα, the SRC1 NR-box region and NRCC-A can not activate
luciferase reporter expression beyond the basal level, indicating NRCC protein does not
contain a transcription activation domain like most other coactivators (Figure 2 C and
Figure 5 B). In the presence of ERα and pM-NRCC-A or NRCC-C, the expression of
49 reporter was significantly enhanced beyond the basal level, consistent with the interaction between ERα and NRCC-A in Co-IP assays (Figure 2 D).
NRCC-A enhances ERα transcriptional activity in mammalian cells
We have demonstrated that NRCC associates with ERα in mammalian cells. To further test the hypothesis that NRCC proteins function as nuclear receptor coactivators, we examined the effect of NRCC-A and NRCC-C on nuclear receptor mediated transcriptional activity. Transient co-transfection experiments were conducted in
Ishikawa cells that express low levels of NRCC mRNA, but do not have detectible ER expression (as reflected in our ERE-luc reporter assays). In an ERα ERE-luc reporter assay, E2 treatment led to a 3-fold activation, which was further increased by 50% in a dose dependent fashion with the addition of NRCC-A (Figure 3A). We observed the same enhancement in COS-7 cells (data not shown). We then tested whether NRCC-C can enhance ERα transcriptional activity in the reporter assay. Little transcriptional enhancement was observed with NRCC-C, indicating the N-terminus of NRCC-A is important for the coactivation function (Figure 3 D). Coactivation by NRCC-A was also seen with AR (Figure 3 C). However, NRCC-A did not enhance ERβ transcriptional activity in the similar assay (Figure 3 B).
We then tested whether NRCC-A itself could act as a transcriptional activator. NRCCs were fused with GAL4 DBD and transfected into Ishikawa cells or Hela cells. The transcription level of a luciferase reporter containing 5 GAL4 binding sites at the promoter region was measured by luciferase assay. In comparison with GAL4-DBD and
50 using GAL4-SRC1-NR-box and ERα two-hybrid as the positive control, NRCC-A and
NRCC-C did not activate Gal4-luc reporter, indicating that they do not have an intrinsic transcriptional activation domain (Figure 5 B).
NRCC-A is a nuclear protein
NRCC-A was predicted to be a nuclear protein by several protein localization software
programs, however a nuclear localization signal was not predicted. To determine the
intracellular localization of NRCC-A, an immunoflorescent assay was performed with
Hela cells transiently transfected with Flag-NRCC-A. As predicted, NRCC-A was
abundantly present in the nuclei of transfected Hela cells. Some NRCC-A was also
detected in cytosol (Figure 4 A). No fluorescence signal was detected in no anti-flag
control or untransfected cells subjected to the anti-flag staining (data not shown).
Immunofluorescent assays also revealed a strong overlapping expression pattern of Flag-
NRCC-A and EGFP-ERα in co-transfected Hela cells, which was unaffected by E2
treatment. GFP-ERα, which is exclusively expressed in the cell nucleus, showed
significant nuclear co-localization with NRCC-A (Figure 4B and C).
NRCC-A is required for endogenous ERα transcriptional activity
NRCC-A overexpression experiments described above indicated that NRCC-A enhances
ERα transcriptional activity in a dose dependent fashion. We also showed that ERα and
NRCC-A may interact in the cell nucleus. As an extension to the above studies and to
further examine the physiological role of NRCC, the requirement of NRCC proteins for
ER-mediated transcription was examined using RNA interference to reduce intracellular
51 NRCC level. Flag-NRCC-A expression was selectively reduced by NRCC siRNA, which significantly impaired estrogen-dependent 3xERE-luc reporter activity (Figure 5 A). The scrambled siRNA control showed no effect on ERE-luc activity (data not shown). This result suggested that NRCC-A plays a substantial role in endogenous ERα transcriptional activity.
As a coactivator, NRCC-A could be recruited to endogenous ER target promoters in vivo.
To test whether NRCC-A is involved in transcriptional activation of native ERα target genes, we employed chromatin immunoprecipitation (ChIP) assays to look for ERα- dependent recruitment of NRCC-A to the ERα-binding sites on pS2 and PR promoters in
MCF-7 breast cancer cells. Control ChIP assays using normal IgG produced very weak signal. NRCC-A was detected on pS2 and PR promoters in the presence and absence of
E2 treatment, but not on the outside control region (Figure 5 C).
NRCC-A plays a role in estrogen-dependent and independent cell proliferation
It is well known that estrogen stimulated cell proliferation in mammary epithelium and
ER positive breast cancer cells is mediated by ERα. Since NRCC-A can enhance ER transcriptional activity, we further analyzed the role of NRCC-A in ERα dependent and independent cell proliferation. Our immunofluorescent assay exhibited dividing cells that were NRCC-A and ER double positive even in absence of E2, suggesting that NRCC-A may promote cell proliferation (Figure 6A, B and C). Thymidine incorporation assays were utilized to monitor cell proliferation (as reflected by DNA synthesis) under different conditions. The role of NRCC-A in ER independent cell proliferation was determined in
52 NRCC-A transfected Hela cells. NRCC-A significantly enhanced DNA synthesis
compared to mock transfected Hela cells. Because NRCC RNAi assays indicated a requirement for NRCCs in MCF-7 cells for ER-mediated transcription, we also determined whether the estrogen-dependent growth of MCF-7 cells is affected by the depletion of NRCCs. As expected, estrogen markedly stimulated the growth of non- transfected (data not shown) and scrambled siRNA transfected MCF-7 cells. However,
NRCC siRNA treatment nearly abolished the estrogen-dependent growth of MCF-7 cells, indicating NRCC proteins are essential for ER-dependent cell proliferation and raise the possibility of NRCC as a potential therapeutic target in breast cancer (Figure 6 C).
Discussion:
In the present study, we identified and characterized NRCC-A, a conserved novel ERα
coactivator in vertebrates. NRCC-A interacts with ERα in a ligand independent fashion
and co-localizes with ERα in cell nuclei. The transcriptional regulatory activity of ERα
was enhanced by NRCC-A, but no intrinsic transcription activity was noted for this novel
cofactor. NRCC-A not only is required for endogenous transcriptional activity of ERα,
but also enhances both ER dependent and independent cell proliferation.
Our findings provide several lines of evidence indicating that NRCC-A functions as an
ERα coactivator. First, NRCC was isolated from a mammary gland cDNA library on the
basis of its interaction with the ERα CDEF domain fragment. In addition to ERα, NRCC-
A also interacts with androgen receptor (AR). Furthermore, as with other classical
coactivators, NRCC proteins contain two LXXLL motifs at the C-terminal region of the
53 protein that can mediate coactivator and nuclear receptor interactions. The functional role
of NRCC-A as a coactivator was also supported by our ERE reporter assays which
showed that NRCC-A enhanced ERα transcriptional activity. Moreover, evidence from
our ChIP assay indicated that NRCC-A is involved in ERα endogenous target gene
transcription. NRCC-A was recruited to the pS2 promoter ERE as well as the half-
ERE/Sp1 site on the hPR promoter.
NRCC shows no sequence homology to known coactivators, suggesting it may belong to
a novel class of NR coactivators. Interestingly, it is a conserved protein in vertebrates,
ranging from human to zebra fish. The coiled-coil domain, including an NR-box buried
in the domain, is conserved from human to Xenopus, however, none of the NRCCs have
been characterized before this study.
Although a nuclear localization signal was not predicted for NRCC, its function as a nuclear protein was confirmed by our transient transfection and immunostaining experiments showing NRCC-A primarily resides in cell nuclei. The coiled-coil domain was predicted by ELM and Paircoil2 (McDonnell et al., 2006) from amino acids 211-241 of the NRCC-A sequence. Coiled-coil domains may form homodimers or interact with
other protein-protein interaction domains (Burkhand et al., 2001; Kim et al 2003),
suggesting that NRCC proteins can form homo- or hetero-dimers and/or interact with
other transcriptional factors or coactivators. The central region of NRCC-A, (amino acids
130-182) is 31% homologous to the RhoA-binding domain (RBD) of ROCK-1 (948-
999aa). This RBD mediates the homodimer formation of ROCK-1 (Dvorsky and
54 Ahmadian, 2004; Dvorsky et al., 2004), indicating that the homologous region on NRCCs
may mediate dimer formation. The LXXLL motif is one of the primary characteristics of
nuclear receptor coactivators. Most classical ER coactivators contain one or more NR-
boxes, which mediate the ligand dependent interaction with ER (Heery et al. 1997). Like other coactivators, NRCC-A contains two NR-boxes, among which the first one is conserved from human to Xenopus. NRCC protein can bind to ER in the absence or presence of ligand, implying the binding of NRCC with ERα may not be solely dependent on the NR-boxes.
NRCC-A interacts with ER and enhances both ligand-dependent and independent ERα
transcriptional activity (Figure 3 A). However, the enhancement is more robust at 12-16
hours after the E2 treatment, suggesting a possible role for NRCC-A in facilitating the
interaction of ERα and other coactivators in the transcriptional complex, or dissociating
corepressors bound to ERα. Unliganded ER bound to NCoR repressor complex (Perissi et
al., 2004; Rosenfeld et al., 2006). Over expression of NRCC-A may trigger the release of
NCoR complex and allow the p160 family members to interact with ER, thereby
activating transcription in the absence of ligand. It is reported that some ligand
dependent corepressors, such as ligand-dependent corepressor (LCoR) and repressor of
estrogen receptor activity (REA), binds ERα in the presence of E2 (Fernandes et al., 2003;
Mussi et al., 2006; Park et al., 2005). LCoR inhibited ERα transcriptional activity in a
dose-dependent fashion by recruiting corepressors HDAC3 and CtBP to ERα. REA
directly compete with p160 coactivators for binding to ER and can recruit class I and II
HDACs (Kurtev et al., 2004; Montano et al., 1999). However, it is not clear how was the
55 ligand-dependent repression is overcome in vivo. The presence of NRCC-A could
counteract the repression by disrupting the interaction of corepressor complex with ERα.
CoCoA, a secondary coactivator binds to Grip1 with its coiled-coil domain and enhances
nuclear receptor transcriptional activity (Kim et al. 2003). Our data cannot rule out the
possibility that NRCC-A interacts with other coactivators through the coiled-coil domain to form a larger receptor/coactivator complex.
Like most NR cofactors, NRCC is expressed in various cells and tissues. The mRNA of
NRCC-A mouse homolog, predicted to code a 303aa protein, was detected in mammary
tumors of 5-month-old MMTV-Wnt-1 transgenic mice (GenBank: AAH24403). Our
result showed that NRCC is required for ER-dependent MCF-7 cell proliferation,
suggesting a possible role of NRCC in cancer development. However, it is not clear how
NRCC-A is involved in cell proliferation. It will be essential to identify other proteins
that interact with NRCC-A using Co-IP and mass spectrometry.
56 Materials and Methods:
Plasmids and antibodies
The cDNA of NRCC (GenBank accession # BC043256) was purchased from Open
Biosystem (Huntsville, AL). NRCC-A，NRCC-B and NRCC-C cDNA were subcloned
into p3XFlag-7.1 expression vector (Sigma) for Flag-tagged fusion protein and pM
(Clontech) for N-terminal Gal4 DBD fusion protein. pSG5AR (Link et al. 2005) and pM-
SRC1-NRbox plasmid containing the nuclear receptor domain of SRC-1 (amino acids
629 to 831) are kind gifts from Dr. Karen Knudsen’s lab. pCMV5-ERα and p3XERE-luc
reporter were provided by Dr. Benita Katzenellenbogen (University of Illinois. Anti-Flag
M2 antibody is from Sigma (Product Code F1804). Anti- ER H184 polyclonal antibody is
from Santa Cruz (Cat # sc-7207). Goat-anti-rabbit secondary antibody (Cat # 31460) and
Goat-anti-mouse secondary antibody (Cat # 31430) are from Pierce.
Cell culture and transient transfection.
Ishikawa cells were grown in MEM (Fisher) with 10% fetal bovine serum (Hyclone).
Hela and MCF7 cells were grown in DMEM (Fisher) with 10% fetal bovine serum. The
transfections were carried out using Fugene 6 according to the manufacturer’s protocol.
At 20 h post-transfection, the cells were treated with specific ligands or vehicle (ethanol)
and were harvested after 12-16 h of incubation. The cells were lysed in reporter lysis
buffer (Promega) and assayed for luciferase activity.
Chromatin immunoprecipitation (ChIP)
57 ChIP assay was performed as previously described (Shang Y et al. 2000). The PCR primers used were as follows: pS2 Forward: 5’TTA GCT TAG GCC TAG ACG GAA TGG; pS2 BACKWARD: GAC GAC ATG TGG TGA GGT CAT CTT-3’ pS2 OUTSIDE Forward: GGG GCT GTT TTC CTG TGT TA-3’ pS2 OUTSIDE BACKWARD: CAT TTG GGC CTA TCT GGA TG-3’
PR Forward: AAA GGG GAG TCC AGT CGT CA-3’
PR BACKWARD: CTG GTCCTGCGTCTTTTCGT-3’
PR OUTSIDE Forward: GGA AGG TTA GAG GAA GAA TG-3’
PR OUTSIDE BACKWARD: CCT TTG CAC CTT TGT TGA GA-3’
Thymidine incorporation
The assay was performed in Hela and MCF7 cells as described (Singleton DS et al. 2002).
Reverse transcription-PCR
For RT-PCR, total RNA was extracted and purified from cells with Trizol (Invitrogen).
Reverse Transcription reactions were carried out with the Retroscript kit (Ambion Cat#
1710), following the manufacturer’s protocol. The resulting products ere subjected to
PCR amplification with the following NRCC primers: Forward: 5’ GCT GAA AAA
GCC AAA TTG GTG 3’and Backward: 5’ GCT GGC TAC AGC TTC TCC ACT 3’.
RNA interference
NRCC siRNA (Dharmacon, Smart mix) or the NRCC siRNA and 3XERE-luc reporter plasmid were introduced into MCF7 cells cultured in 96-well plates by using SilentFect
58 (BioRad Cat# 170-3360), following the manufacturer’s protocol. Luciferase activities were assayed with Luciferase Reporter Assay System (Promega).
Co-immunoprecipitation and Western Blot
Co-IP was performed according to the standard protocol provided by Santa Cruz
Biotechnology. Western Blot was performed according to the standard protocol provided by Sigma for Anti-Flag M2 antibody. Nitrocellulose membrane was from Bio-RAD (Cat#
162-0094). ECL kit is from Amersham Biosciences (cat # 1059250)
Immunofluorescent assay
Immunofluorescent assay was performed according to the standard protocol provided by
Sigma for Anti-Flag M2 antibody
Digital photography
The pictures were photographed with a Zeiss LSM510 confocal microscope (Carl Zeiss
Co. NY) with 63X Zeiss objective lens.
Mammalian hybrid assays
Mammalian hybrid assays were performed as described (Martini et al., 2000), except that a pM-SRC1 construct and pCMV5ERα were co-transfected with 4XGal4-luc reporter into the Ishikawa cells. 16h after the transfection, the cells were treated with E2 for 24h and subjected to luciferase assay.
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62
Figures:
Figure 1
A NRCC-A 1 307 NRCC-B 245 NRCC-C M 232
MAEG NR-Box Coiled-Coil
B
Chimpanzee TAA-EKNQDFFMPT K ESSDSAVVLPSTPAAPPQSN SS YTNSSRKQSTPM ALLFRER RKTR 244 Rhesus AFFMEG-EKNQD T K ESSSDSAVVLPSTPAAPPQSN SS YTNSSRKQSTPM ALLRER RKTRS 60 HUMAN SFL-EKNQDFFMPT K ESSDSAVVLPSTPAAPPQSN SS YTNSSRKQSTPM ALLFRER RKTR 122 Dog RALGEVSQDCVFKMPESSDPAVVSPPAAST QCSSPP PPR SSSRKQSTPM ALLFRER RKTR 300 Mouse NSPP PPTS PAAPQPRENSPP PPTS PAAPQPRENSPP PHSNSSGKQPLSGTPLAKER KK RS 139 .. . : ...... *::**. .*.** ..** ***:*.* :***:*:*
Chimpanzee SFNSSYNVVKRLVFK ESEENDQT SEKPASSTEENCLFFIFLLE QES KH DSE EENTN KNT K 304 Rhesus SFNSCYNVVKRLVFK ESEENDQT SDKPASSTEENCLFFIFLLE QES KH DSE EENTN KNT K 120 HUMAN SFNSSYNVVKRLVFK ESEENDQT SEKPASSTEENCLFFIFLLE QES KH DSE EENTN KNT K 182 Dog SFNSCYSAVKRLVK ENEENDQTFPEEPILFFIS-- EENC D QEN KH DSPFKESTCLFKNT K 358 Mouse SSHSFCSVVKRMVLPK ENDENNETSE GESSKEENCSKALPQES KNKDSE GEKSSEEK--- 196 * :* ..***:***.:**::*:.: **** . **.:*: ** *.: ::
Chimpanzee NNCLVLESQS DSGSCSALQNEFVNEKKLP QRLANDEKKKLV QQV EKEDLLRRLKKLV MYR 364 Rhesus NSCLVLESQS HSGSCSAL QNEFMNENLPKQRLANDEKKKLV QQV EKEDLLRRLKKLV MYR 180 HUMAN NNCLVLESQS DSGSCSALQNEFVSEKKLP QRLAAN EKKKLV QQV EKEDLLRRLKKLV MYR 242 Dog SSCIALLESKS DTESRSD QSDFVNEDLLASKQG SEERKKLV QIEEKEDLLRRLKKLV MYR 418 Mouse --NTCESKSSDTGSSNALPKESENAIIR-EKKLIQEKRRLI QVEEKEDLLRRLKKLV MYR 253 ..***:* .: * . * .: . : : *. *: :*::*::****************
Chimpanzee SKNDLLSQ QLLIKKWRSCSQLLLYELQSAVSE-ENKKLLS TQLIDHCGLDDKLLHYNRSE 423 Rhesus SKNDLLSQ KVLIKKWRSCSQLLLYELQSAVSE-ENKKLLS TQLIDHYGLDDKLLHYNRSE 239 HUMAN SKNDLLSQ QLLIKKWRSCSQLLLYELQSAVSE-ENKKLLS TQLIDHYGLDDKLLHYNRSE 301 Dog SKNDLLSQ QQLLI KRW SCSQLLLYELQSAMSE-ENKKLLS TQLIDHCGLDDKLLHYNRNE 477 Mouse IVLKND TEENLIKKWRKCGQRLLCELQSIMSEDEDEKLLTTEDLI FIYG DDNLLHYNRSE 313 ***:::*: **:***.*.* ** **** :** *::**:**:***. *:**:******.*
Chimpanzee EEFIDV 429 Rhesus EEFIDV 245 HUMAN EEFIDV 307 Dog EEFMGV 483 Mouse EEFVTG 319 *** .*
C 1 2 3 4 5 6 7
63
Figure 2
A 1 2 3
B IP: H184 anti-ER 5% input E2 - + - +
IB: Flag-NRCC-A
IB: ERa
IP: M2 anti-Flag
IB: ERa
Flag-NRCC-A C c 30000 E2 25000 20000 15000 RLU 10000 5000 0 ERa + - + + NRCC-A + - - - SRC1 - + + -
64
Figure 3
A hERa B hERß
c 1200 1800 c E2 1600 E2 1000 1400 1200 800 1000 600 800 400
1000 X RLU 600 1000 X RLU X 1000 400 200 200 0 0 0520 030 NRCC-A (ng) NRCC-A (ng) CD hAR hERa c 5000 c 1000 4500 DHT 900 E2 4000 800 3500 700 3000 600 2500 500 2000 400 100 xRLU
1000 X RLU X 1000 1500 300 1000 200 500 100 0 0 030 020 NRCC-A (ng) NRCC-C (ng)
65
Figure 4
66
Figure 5
AB
2500 c 30000 c E2 E2 2000 25000 20000 1500 15000 RLU 1000 RLU 10000 500 5000 0 0 02040 NRCC-A Vector Control NRCC siRNA (nM)
C M2 Input IgG pS2 promoter 1 2 3 4 5 6 E2 - + - + - + ERE pS2 ERE
-3200 -404 -392 Outside
PR promoter
Sp1 PR Sp1 -3500 BA+1282 +1306 Outside
67 Figure 6
NRCC-A ER-GFP Merged ABC
DE
c 1000 c 8000 serum 900 E2 7000 800 6000 700 5000 600 4000 500 CPM CPM 3000 400 300 2000 200 1000 100 0 0 Control NRCC-A Control NRCC
RNAi
68
Figure legends:
Figure 1 NRCC protein structure and mRNA expression in different tissues and cells.
(A) Schematic representation of NRCC protein isoforms. The NR-box and Coiled-coil
domain are indicated. Existence of the Coiled-coil domain was predicted using ELM
(www.expasy.com). MAEG indicated the first 4 amino acid of NRCC-B that is not present in the NRCC-A sequence. (B) NRCC-A is conserved in mammals. The multiple sequence alignment was performed with EMBL-EBI ClustalW online software on the following website: http://www.ebi.ac.uk/clustalw/ The homologies of human NRCC-A
with other mammalian species are shown, with asterisks indicating positions of identical
amino acids, dot and colon indicating the positions of similar amino acids. LXXLL
motifs are indicted by rectangle boxes. C) NRCC-A transcripts are widely expressed.
Total RNA was isolated from the indicated cell lines and subjected to RT-PCR with
NRCC-A primers. Human fetal brain and skeleton muscle cDNA was purchased.
1.Ishikawa cells 2.C4-12 cells; 3. MCF-7 cells; 4. low mass ladder (Invitrogen); 5.
Human Skeleton Muscle cDNA; 6. Human Fetal Brain cDNA; 7. Mouse embryonic
fiberblast (MEF) cells. C4-12 cells and MCF7 cells are human breast cancer cells.
Ishikawa cells are human endometrial cancer cells. The human NRCC-A primers do not
amplify mouse NRCC-A.
Figure 2 NRCC-A interacted with ERα in mammalian cells. A) Flag-NRCC-A protein
was detected in Ishikawa cells. Flag-NRCC-A construct was transfected into Ishikawa
cells and Western Blot was used to detect the expression of Flag-NRCC-A, using M2
anti-Flag antibody. A 37 kD fragment was detected (lane 1 and 2). No signal was
69 detected in monk transfected Ishikawa cells (lane 3). B) Flag-NRCC-A and ER were co- transfected into Ishikawa cells. Co-IP experiments were performed with anti-ERα and anti-Flag antibodies in Ishikawa cells at the presence and absence of E2. C) The interaction of ERα and NRCC-A was also analyzed with mammalian two-hybrid experiments in Ishikawa cells. pM vector, pM-NRCC-A (NRCC-A) and pM-SRC1 were used as baits. SRC1 NR-box (SRC1) and ERα was used as positive control.
Figure 3 NRCC-A enhances ERα and AR transcriptional activity.
(A, B and C) NRCC-A enhances ERα and AR transcriptional activity but not ERβ. (D)
NRCC-C does not affect ERα transcriptional activity. Ishikawa cells were co-transfected with nuclear receptors (ERα, ERβ and AR) and their respective reporter genes (3xERE- luc and ARE-luc), and indicated amount of p3xFlag-NRCC-A (A, B and C) or p3xFlag-
NRCC-C (D). The cells were treated with vehicle or 1 nM E2 or DHT. Transcriptional activity was measured via luciferase assay. All experiments were performed multiple times with triplicate samples. The error bars indicated standard deviations.
Figure 4 NRCC-A and ERα co-localized in cell nucleus. Immunoflourescent staining was performed on Hela cells co-transfected with Flag-NRCC-A and GFP-ERα. 24 hours after the transfection, cells were treated with E2 or vehicle overnight. The cells were fixed and stained with M2 anti-flag antibody. Immunostained cells were photographed using a
Zeiss LSM510 confocal microscope with 63X Zeiss objective. A. Flag-NRCC-A; B.
GFP-ERα; C. Merged images of Flag-NRCC-A and GFP-ERα. Scale bar = 20 μm
70 Figure 5 NRCC-A is a nuclear receptor coactivator. (A) NRCC-A is required for endogenous ER transcriptional activity. MCF-7 cells were co-transfected with indicated amount of NRCC siRNA and 3xERE-luc reporter or scrambled siRNA and 3xERE-luc reporter. 24 h after the transfection, The cells were treated with 1 nM E2 or vehicle for 24 h. Transcriptional activity was measured via luciferase assay. (B) NRCC-A does not contain intrinsic transcriptional activity. Mammalian one-hybrid experiment was
performed in Ishikawa cells. GAL4DBD (Vector), GAL4DBD-NRCC-A (NRCC-A), and
GAL4DBD-SRC1NR-box plus ER (Control) were co-transfected with 4XGal4-luc
reporter. SRC1-ER two-hybrid was used as positive control. The level of transactivation
was represented by the relative luciferase activity. (C) NRCC-A was recruited to the
promoter of endogenous ER target gene promoters. The in vivo binding of NRCC-A to
pS2 and PR promoter were examined by the ChIP assay. Flag-NRCC-A was transiently
transfected into MCF7 cells. Soluble chromatin was prepared from the cells treated with
1 nM E2 (+) for 1 h or untreated (-) and immunoprecipited with M2 antibody. Co-
precipitated DNA was amplified using primer that flank the ERE in the pS2 promoter
region or half ERE and Sp1 site in the PR promoter. The presence of total pS2 and PR
promoter DNA in the soluble chromatin prior to immunoprecipitation was included as
input.
Figure 6 NRCC-A promotes cancer cell proliferation. Immunoflorescent staining (A, B
and C) and thymidine incorporation (D and E) were used to determine the role of NRCC-
A in cell proliferation. Flag-NRCC-A (A) and ER-GFP (B) were detected in Hela cells.
Some dividing cells were NRCC-A and ER double positive (arrow in C). Scale bar = 20
71 μm. In thymidine incorporation assays, NRCC-A cDNA was transfected into Hela cells
(D) to determine the impact of NRCC-A on hormone independent cell proliferation.
NRCC siRNA was transfected into MCF-7 to determine the effect of endogenous NRCC protein on ER dependent cell proliferation.
72 Chapter III
BIOLOGICAL SCIENCES: Developmental Biology
Estrogen receptor α expression in the mammary epithelium is required for ductal
and alveolar morphogenesis in pubertal, pregnant and lactating mice
Yuxin Feng1, David Manka1, Kay-Uwe Wagner2 and Sohaib Khan1
1Department of Cell and Cancer Biology, Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, Cincinnati, Ohio
2Eppley Institute for Research in Cancer and Allied Diseases and the Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska
Address correspondence to: Sohaib Khan, PhD Department of Cell and Cancer Biology University of Cincinnati, College of Medicine Vontz Center for Molecular Studies 3125 Eden Avenue Cincinnati, OH 45267-0521 Tel. 513-558-7224 Fax. 513-558-2445 Email: [email protected]
73 Abstract
The estrogen receptor α (ERα) is a critical transcription factor that regulates epithelial
cell proliferation and ductal morphogenesis during postnatal mammary gland
development. Tissue recombination and transplantation studies using the first generation
of ERα knockout (ERKO) mice suggested that this steroid hormone receptor is required
in the mammary stroma that subsequently exerts its effect on the epithelium through
additional paracrine signaling events. A more detailed analysis revealed that ERKO
mice produce a truncated ERα protein with detectable transactivation activity, and it is
likely that this functional ERα variant has masked the biological significance of this
steroid receptor in the mammary epithelium. Moreover, the ERα transplantation model
is less suitable to study the loss-of-function of this gene throughout mammogenesis. In this new report, we describe the generation of Cre-lox-based conditional knockouts of the
ERα gene to study the biological function of this steroid receptor in the epithelial
compartment at defined stages of mammary gland development. The MMTV-Cre-
mediated, epithelial-specific ablation of exon 3 of the ERα gene in virgin mice severely
impaired ductal elongation and side branching. This conditional knockout resulted in a
complete ablation of the ERα protein, and the progesterone receptor (PR), whose
expression is under the control of ERα, was largely absent. WAP-Cre-mediated deletion
of ERα during successive gestation cycles resulted in a loss of ductal side-branching and
lobuloalveolar structures, ductal dilation, and decreased proliferation of alveolar
progenitors. These abnormalities compromised milk production and led to
malnourishment of the offspring by the second pregnancy. These observations suggest
74 that ERα expression in the mammary epithelium is essential for normal ductal morphogenesis during puberty and for alveologenesis during pregnancy and lactation.
75 Introduction
Estrogen receptor (ER) is a transcription factor that regulates the genetic program
of cell cycle progression and growth in healthy and cancerous mammary glands in
response to circulating ovarian hormones. Of the two receptor forms, ERα and ERβ, the
former is considered the primary receptor for mammary gland development and function.
The mammary gland is unique compared to every other organ of the body in that the bulk
of its development and differentiation occurs postnatally. Following birth and until
puberty, the mammary gland is dormant and rudimentary. At puberty, the mammary
gland develops rapidly in response to changes in circulating hormone levels. Terminal
end buds (TEBs) that are composed of multiple layers of cuboidal epithelial cells begin to
form at the termini of primitive ducts. The TEBs invade the fat pad and give rise to a
branching network of ducts that end at the edge of the mammary fat pad. Growth of the
ductal network ceases at this stage until further stimulation during pregnancy.
Seminal studies by Korach and colleagues, indicated a critical role for ERα in mammary development in which the ERα gene was disrupted by the insertion of a neomycin resistance gene (neo) into the first coding exon (Bocchinfuso et al., 2000;
Korach et al., 1996). Mice carrying this insertion are hypomorphic for ERα in that substantial ERα function is retained (Kos et al., 2002). Circulating prolactin (PRL) levels were reduced in hypomorphic ERα females and there was a lack of mammary gland development beyond the prepubertal stage (Bocchinfuso et al., 2000). Restoration of PRL levels by pituitary isograft normalized mammary gland development, which could be prevented by ovariectomy. Likewise, exogenous estradiol and progesterone induced normal ductal elongation and TEB formation in hypomorphic ERα females.
76 Thus, the observed phenotype was in part due to abnormal pituitary and ovarian hormone levels in the animals, whereas ERα function was largely retained in the mammary gland.
The mammary gland consists of multiple cell types including luminal and basal
epithelial cells, stromal cells, adipocytes, and vascular endothelial and smooth muscle
cells. In the mammary glands of rodents, both the epithelium and stroma express ERα
(Edery et al., 1984; Haslam and Nummy, 1992; Haslam and Shyamala, 1981). In an
effort to dissect the complex paracrine and autocrine regulation of mammary gland
development by estrogen, Cunha and colleagues pioneered a novel technique of tissue
recombination in vivo consisting various combinations of stromal and epithelial
compartments from hypomorphic ERα and wild-type mice (Cunha et al., 1997). The results from this and subsequent studies (Mueller et al., 2002) suggested that stromal
ERα was necessary – whereas epithelial ERα was dispensable – for mammary gland development. This suggested role of ERα in the mammary gland of rodents appears to contradict the finding that tamoxifen treatment of breast cancer patients targets ERα in the cancer cells themselves and not the stroma of the breast. This issue is further substantiated by the fact that these targeted therapies are effective in the treatment of metastatic breast cancer cells which are epithelial in origin and interact with stromal cells of distant organs.
A more recent ERα knockout (αERKO) mouse model has been developed in
which exon 3 of the ERα gene was deleted without any detectable expression of ERα
transcript (Dupont et al., 2000b). Mammary glands from genetic αERKO mice were
normal prior to puberty. After the onset of puberty, however, TEBs remained absent and
ducts failed to invade into the fatpad beyond the nipple area (Mallepell et al., 2006). In
77 neither the ERα hypomorphic nor the αERKO mouse does the mammary gland develop
beyond puberty. Although it was not reported, a reduction in circulating ovarian and
pituitary hormones is likely in the αERKO mouse, similar to the ERα hypomorphic
mouse (Bocchinfuso et al., 2000; Couse and Korach, 1999).
In this study, we sought to dissect the role of epithelial versus stromal ERα in mammary gland development, maturation and lactation. We found that postnatal deletion of epithelial ERα arrests mammary gland development at the prepubertal stage.
Strikingly, the deletion of mammary epithelial ERα during late pregnancy revealed that
ERα is critically important for alveologensis and lactation during repeated gestational
cycles.
78 Results
A targeting vector was generated from a mouse ERα genomic clone in which
exon 3 was flanked by loxP recombination sites and electroporated into 129/SvOla
embryonic stem (ES) cells. The targeted ERα allele was confirmed with Southern blot
analysis of BamHI –digested ES cell genomic DNA, using 5’ and 3’ external probes (Fig.
1A). The loxP-flanked (floxed) pGK-Neo cassette was deleted from the ES clones and
identified by 5’ and 3’ Southern blot and genotyping PCR (Supporting Fig. 6A and 6B).
Targeted ES cells were injected into C57BL/6 blastocysts and returned to a
pseudopregnant host of the same strain. Chimeric males were obtained that transmitted
the mutation through crosses with C57BL/6 females, producing heterozygous ERαfl/+ mice (mice bearing one floxed allele in which exon 3 is flanked by loxP sites). Matings of ERαfl/+ yielded ERαfl/fl mice that were genotyped using PCR on genomic DNA of tail biopsies (Supporting Fig. 6C).
In order to study the role of ERα in the epithelium during different stages of
mammary gland development, the ERαfl/fl mice were bred with the well-characterized
MMTV-Cre mice (Wagner et al., 2003; Wagner et al., 2001; Wagner et al., 1997), thus generating the first ERα conditional knockout model (MMTV-Cre ERαfl/fl or MMTV-
ERKO). As demonstrated previously, the expression of the MMTV-Cre transgene is
limited to epithelial cells in the mammary gland and is not expressed in the mammary
stroma (Wagner et al., 2001). MMTV-ERKO mice were viable and developed to adulthood, but the mammary ductal outgrowth arrested at the pre-pubertal stage (Fig. 2).
At the age of 6 months, the ductal network of the MMTV-ERKO mice had scarcely invaded into the fat pad and side-branching was minimal (Figure 2A). Few terminal end
79 buds (TEBs) were detected in MMTV-ERKO mice (Figure 2C & 2E). Age-matched
ERαfl/fl control females exhibited abundant TEBs, extensive ductal branching and normal
fat pad invasion (Figure 2B, 2D and 2F). These observations clearly indicate that ERα is
essential in the epithelial compartment of the murine mammary gland and not the stromal
compartment as previously suggested (Cunha et al., 1997; Mueller et al., 2002).
Next we bred WAP-Cre transgenic mice (Wagner et al., 2003; Wagner et al.,
2001; Wagner et al., 1997) with the ERαfl/fl mice to specifically ablate the ERα gene in
the mammary epithelium during late pregnancy and lactation (WAP-Cre / ERαfl/fl or
WAP-ERKO mice). The expression of the WAP-Cre transgene is largely limited to epithelial cells located at duct termini and within developing alveoli (Wagner et al., 2002;
Wagner et al., 1997). As expected, ductal morphogenesis was normal in nulliparous
(virgin) WAP-ERKO mice and equivalent to ERαfl/fl control females (data not shown).
As expected, deletion of the ERα floxed locus was not detected in nulliparous WAP-
ERKO mice (Fig. 1B, lane 3). Using PCR, we confirmed the deletion of ERα in WAP-
ERKO mice following pregnancy (Fig 1B, lane 4). Excision of ERα in WAP-ERKO
mammary glands was first detected in primiparous females at lactation day 13.5 (L13.5).
But even during the second lactation, intact ERαfl/fl alleles were still detectable in WAP-
ERKO females (data not shown), suggesting that a) the WAP-Cre transgene exhibits a
mosaic expression profile, and b) there is a negative selection pressure against ERα knockout cells. No ERα deletion was detected by genotyping PCR in other ERα- expressing organs including uterus, ovary, skin and brain collected from WAP-ERKO females, verifying the specific expression of Cre recombinase in the mammary glands of
WAP-Cre mice (data not shown). Hypomorphic αERKO mice are characterized by high
80 circulating level of E and reduced level of P as well as PRL, all of which are required for
normal mammary gland development, differentiation and lactation (Bocchinfuso et al.,
2000; Couse et al., 1995). Using commercial ELISA kits, we found that serum estradiol
and P levels are within the normal physiological range in both virgin WAP-Cre / ERαfl/fl and pregnant WAP-ERKO mice compared to ERαfl/fl mice (n=3 females per group) (Data
not shown). In a cell-based assay, in which the proliferation of PRL-dependent
mammary epithelial cells is quantified (Spragg et al.), we found that serum prolactin
levels were also normal in the conditional knockouts (data not shown).
To determine the impact of ERα conditional deletion on mammary gland
development we examined the inguinal mammary glands (#4) from WAP-ERKO and
ERαfl/fl females at various stages of successive pregnancy and lactation cycles. During
the first lactation, a mosaic phenotype was detected in the mammary glands of WAP-
ERKO females. Many secretory alveoli were of normal size. However, alveolar growth
was less extensive in various parts of the mammary gland of lactating WAP-ERKO
females, and we also observed a mild defect in tertiary ductal branching during the first
pregnancy and lactation (Supporting Fig. 7). In contrast, ductal structures were obscured
and not directly visible due to the normal alveolar expansion in ERαfl/fl control mice
during the first and also the second pregnancy (Fig. 3A & B). However, during the
second pregnancy cycle WAP-ERKO females exhibited aberrantly dilated ducts with few
side-branches that were filled with milk-like secretions (Fig. 3C & D). We observed strikingly fewer lobular alveoli in WAP-ERKO mice compared to ERαfl/fl control females
(Fig. 3C). The diameters of engorged WAP-ERKO ducts (Figure 3D) were an order of
magnitude larger than ERαfl/fl control ducts (Figure 3B).
81 Unlike transplant models that are not suitable for lactation studies due to the lack
of a nipple connection, the WAP-Cre-based, mammary-specific knockout of ERα
allowed us to assess the importance of ERα for the growing offspring. Using the
conditional knockout mice, we observed that ERα deficiency had a profound impact on
the normal growth of the offspring. About one third of the pups nursed by WAP-ERKO
mothers were malnourished during the 1st lactation period. The average body weight of
21-day-old pups was about 17% less than that of pups from similar-sized litters nursed by
ERαfl/fl females (10 g versus 12 g, n=8, P<0.001). The growth retardation of the pups
was more severe during the second lactation. At this time, the average weight of 6-day-
old pups nursing on a WAP-ERKO mother was about 50% that of control pups nursing
on ERαfl/fl dams (Fig. 4). The growth retardation of the offspring from WAP-ERKO
females was fully rescued by fostering them with ERαfl/fl mothers (Fig. 4). Milk
harvested from WAP-ERKO females contained normal concentrations of whey acidic
protein (Supporting Fig. 8), which is critical for offspring nourishment (Triplett et al.,
2005). This suggests a defect in milk production and delivery attributable to abnormal
glandular architecture rather than production of inferior-quality milk in WAP-ERKO
nursing females. Accordingly, the volume of milk harvested from nursing WAP-ERKO
females during the 2nd lactation cycle was less than half that of ERαfl/fl females (n = 2 per
group, data not shown).
Based on the proposed role of WAP-Cre-expressing, parity-induced mammary epithelial cells (PI-MECs) that survive the first gestation cycle and function as alveolar progenitors during subsequent pregnancies, we reasoned that progressive loss of ERα- and downstream PR-mediated signaling in the mammary gland with successive
82 pregnancies was responsible for the observed mammary gland defects and offspring
malnourishment. Progressive loss of ERα was verified by immunohistochemical staining
of WAP-ERKO mammary glands during the second lactation (Fig. 5). While epithelial
nuclear ERα was nearly undetectable in the abnormally enlarged mammary ducts of
biparous WAP-ERKO mice (Fig. 5A), ERα expression in stromal cells was abundant
(Supporting Figure 10). Accordingly, expression of PR, which is a transcriptional target
of ERα in mammary epithelial cells, was similarly absent (Fig. 5B). The loss of ERα
and PR only occurred following lactation, as WAP-Cre / ERαfl/fl virgin mice displayed
robust nuclear staining throughout the mammary gland luminal epithelium (Supporting
Fig. 9A and 9B). Multiple nuclei of ductal and lobular epithelial cells of control ERαfl/fl
expressed ERα and PR in the first day of the second lactation (Fig. 5C and 5D). The expression of ERα in uteri of WAP-ERαfl/fl females (2-month-old virgin and 5-month-old adult in first involution) were also analyzed, the expression patterns of ERα in WAP-
ERαfl/fl were similar to age matched ERαfl/fl controls (data not shown), confirming the
specificity of Cre recombinase expression. Next, we analyzed the expression of the proliferating cell nuclear antigen (PCNA) to determine the possible cause of the ductal dilation and loss of secretory alveoli. By the 1st day of the 2nd lactation, ductal and
alveolar epithelial cells from control ERαfl/fl mammary glands robustly expressed PCNA.
In contrast, PCNA expression was diminished in the enlarged ducts and alveoli of WAP-
ERKO mammary glands. The number of nuclei of mammary epithelial cells in lactating
WAP-ERKO mice that were PCNA-positive was about half of that observed in control
ERαfl/fl mammary glands (data not shown). Thus, the dilation was not due to increased
83 proliferation of ductal epithelial cells, while the loss of alveoli may be due in part to impaired ERα-dependent proliferation of luminal epithelial cells.
84 Discussion
We found that progressive loss of ERα in mouse mammary glands during gestational cycles results in loss of lobuloalveoli, impaired ductal side-branching and inadequate milk delivery. MMTV-Cre-mediated excision of ERα occurs in multiple organs shortly after birth. The resulting mammary gland phenotype of arrested ductal growth at the prepubertal stage was similar to the conventional ERα knockout (Mallepell et al., 2006). Our data indicates that early and complete loss of ERα throughout the mammary epithelium prevents the formation of TEBs and severely impairs ductal elongation. Previously published studies that utilized tissue recombination and the ERα hypomorphic model (Cunha et al., 1997; Mueller et al., 2002) suggested that ERα was dispensable in the mammary epithelium. The authors proposed a predominant role of
ERα signaling in the stroma, which created much doubt in the scientific community about the legitimacy of mice to model ERα-based breast cancer prevention since the mammary stroma in humans express little ERα. Then again, our findings in the Cre/lox- based conditional knockout model of the estrogen receptor clearly indicate that ERα is essential in the epithelial compartment of the murine mammary gland. Therefore, our proposed model, which emphasizes the significance of ERα signaling in the epithelium, might also suggest that tamoxifen has a direct effect on the growth of premalignant lesions in selected ERα-positive murine mammary cancer models. Recent studies by
Medina and colleagues demonstrate that tamoxifen had a profound impact on the prevention of mammary tumorigenesis in the p53 knockout transplant model (Medina et al., 2005). The newly-designed ERα-expressing breast cancer models in combination
85 with the ERα conditional knockout model will be invaluable for studying estrogen
receptor signaling in premalignant and cancerous lesions of the mammary gland.
Essential functions of ERα were primarily associated with ductal elongation
rather than lobuloalveolar formation (Cunha et al., 1997). Recent studies by Mallepell
and colleagues (Mallepell et al., 2006) establish a role for ERα in mammary epithelial
cells during puberty, and these observations are in full agreement with the phenotypic analyses in the MMTV-Cre-based conditional knockout mice. However, the transplant model by Mallepell et al. and the MMTV-ERKO mice provide limited insights into the role of ERα signaling at later stages of mammogenesis. Using WAP-ERKO mice, we
were able to specifically ablate ERα in duct termini and alveolar units after ductal
elongation was completed. Also, these mice permitted us to study the loss of ERα during
multiple pregnancies and lactation cycles. Successive gestational cycles drove a
progressive loss of ERα in the mammary epithelial cells (MECs). In summary, our
observations clearly indicate that ERα signaling is required for ductal elongation.
Furthermore, ERα is equally important for pregnancy-induced tertiary branching and the
proliferation and maintenance of differentiating alveolar cells (i.e. WAP-Cre is first
activated during the second half of pregnancy when alveolar cells assume an advanced
differentiation profile).
Paracrine signaling between neighboring cells within the mammary gland can
compensate for lack of ERα (and thus PR) in specific mammary epithelial subtypes. A
10:1 ratio of ERα-competent to ERα-knockout (ERKO) transplanted MECs reconstituted
normal mammary gland development and induced participation of ERKO MECs in all
epithelial compartments: luminal and basal cells, as well as cap and body cells of TEBs
86 (Mallepell et al., 2006). A 1:1 ratio of PR-competent to PR-deficient transplanted MECs
similarly rescued mammary gland development (Brisken et al., 1998). We have found
that alveolar abnormalities can emerge very early after initiation of ERα excision from
MECs during the first lactation, uncovering a greater stringency for adequate ERα and
PR signaling during reproduction that may have been overlooked or not feasible with previous transplantation approaches. More importantly, our observation of the critical role of ERα and PR during lactation was unexpected since estradiol and P levels decline sharply following birth due to the loss of the corpus luteum.
Expression of the WAP gene continues throughout lactation and the highest
expression is restricted to differentiated luminal mammary epithelial cells (MECs).
Expression declines rapidly with weaning, but a subpopulation of hormonally responsive
alveolar MECs resist apoptosis and survive involution (Wagner et al., 2002; Wagner et al.,
1997). These parity-induced mammary epithelial cells (PI-MECs) are predominantly
located within terminal ducts and alveolar units of involuted mammary glands (i.e. the murine equivalent of terminal duct lobular units in the human breast). Using genetic labeling, it has been shown that PI-MECs are able to self-renew and they serve as alveolar progenitors in successive pregnancies. The increasing penetrance of the abnormal phenotype in multiparous WAP-ERKO females might suggest that PI-MECs critically require intrinsic ERα signaling to numerically expand during successive gestation cycles and that the ablation of ERα can inhibit the capacity of this epithelial subtype to self-renew. Since PI-MECs possess features of multipotent stem cells upon
transplantation into the cleared fat pad of recipient mice (Wagner et al., 2002; Wagner et
al., 1997) and (Matulka et al., submitted), the ERα conditional knockout mice might be a
87 valuable tool to address in future studies the importance of ERα signaling in multipotent progenitors and mammary epithelial stem cells.
88 Materials and Methods
Generation of conditional ERα knockout mice. The mouse ERα genomic clone from
129/sv embryonic stem (ES) cells containing exon 3 of ERα was a kind gift from Jan-
Ǻke Gustafsson of the Karolinska Institute. The 9.2kb BamHI fragment containing exon
3 consists of nucleotides 655 to 845 and amino acids 156-218 and encodes the first zinc finger of the DNA binding domain (White et al., 1987). The floxed pGK-neo cassette, a kind gift from Philips Sanford and Tom Doetschman, University of Cincinnati, was cloned into the Eco47III site, and a loxP site followed by a BamHI site were introduced at the NheI site. An HSV-TK expression cassette was subcloned into the HpaI site as the negative selection marker. The 12.2 kb targeting vector linearized at the PmlI site was electroporated into 129/SvOla ES cells (Figure 1). ES cells containing a targeted ERα allele were identified by 5’ and 3’ outside PCR. The 5’ outside primers were: forward 5’-
AGCAAGGGAAAACAAAAACCTGTGT-3’ and reverse 5’-
AGTCATAGCCGAATAGCCTCTCCAC-3’. The 3’ outside primers were: forward 5’-
CTATCAGGACATAGCGTTGGCTACC-3’ and reverse 5’-
AATGAGAGAGGACCAGCGATCTTAT-3’. Genotyping on tail DNA was performed by PCR using the following primers: forward 5’-TGGGTTGCCCGATAACAATAAC-3’, and reverse 5’-AAGAGATGTAGGGCGGGAAAAG-3’. The size of the amplified DNA fragment was 1280 bp in ERαfl/fl and 1200 bp in wild type (WT) mice as separated on a
1.3% agarose gel. ERαfl/fl mice were bred with transgenic mice expressing the Cre enzyme in the mammary tissue under the control of the mouse mammary tumor virus
(MMTV) long terminal repeat and the whey acidic protein (WAP) gene promoter as described (Wagner et al., 1997). Cre-mediated excision of the ERα gene was verified by
89 genotyping PCR of DNA extracted from tail clips using the same primers described above.
Blood and tissue collection and analyses. Mice were killed in a CO2 chamber according to IACUC and institutional protocol. Blood was collected from the ventricle of the heart with a 23G needle. After clotting at room temperature for two hours, the blood was centrifuged and the serum collected and stored at -80°C. Mammary glands were dissected, mounted on glass slides and fixed and stained in carmine alum solution as described for whole-mount analysis (Bocchinfuso et al., 2000). For immunohistochemical analysis, tissues were fixed overnight in 10% neutralized buffered formalin (Richard-Allan Scientific catalog number 9400-5), processed and embedded in paraffin according to standard procedures by the University of Cincinnati Comparative
Pathology Core Laboratory. H&E staining was performed as previously described
(Manka et al., 2005). Serum estradiol and progesterone levels were measured according to the manufacturer’s instructions (Cayman Chemical, cat. nos 582251 and 582601).
Prolactin levels were determined by a cell-based bioassay as described (Liby et al., 2003).
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining of whey acidic proteins in the milk of lactating ERαfl/fl (+/+) and WAP-ERKO (-/-) females
was performed as described (Triplett et al., 2005).
Immunohistochemistry. Slides were boiled in 1X citrate buffer (pH 6.0) for 20 minutes
for antigen retrieval. Immunostaining with ERα (1:150, sc-542, Santa Cruz) and PR
(1:250, sc538, Santa Cruz) antibodies were performed as described (Cheng et al., 2004).
PCNA (1:1000, sc-56, Santa Cruz) and K18 (1:30, 61028, Progen) IgG were used with
the Histo-mouse-MAX kit (Zymed Lab cat. #: 87-9551) to detect protein expression
90 according to the manufacturer’s instructions. K14 (1:1000, prb-155p, Covance) IgG
staining was performed according to the standard protocol from the manufacturer.
Biotinylated secondary antibody (goat anti-rabbit IgG) was from Zymed Lab and the
avidin-biotin kit was obtained from Vector Lab. Digital photomicrographs were acquired
with a Nikon Microphot – FXA microscope and SPOT 4.0.9 for Windows (Contact
Diagnostic Instruments, Sterling Heights, MI).
Statistical analysis. Data were compared by a one-way analysis of variance (ANOVA)
followed by a one-tailed Student’s t-test to evaluate levels of significance at 95% confidence. Differences were determined to be statistically significant when p<0.01 unless otherwise noted.
Acknowledgements
We thank Dr. Keith Stringer (Cincinnati Children’s Hospital Medical Center) for
assistance with ERα and PR staining; Drs. Eric Hugo and Nira Ben-Jonathan (University of Cincinnati) for the prolactin bioassay; and Dr. Nelson Horseman (University of
Cincinnati) for helpful discussions. This work was supported by NIH: CA72039;
HD297731; T32 HD07463 and DOD BC981038
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94 Figures:
Figure 1
95
Figure 2
96
Figure 3
97
Figure 4
98
Figure 5
99
Figure 6
100
Figure 7
101
Figure 8
102
Figure 9
103
Figure 10
104
Figure legends
Fig. 1. Targeted and conditional disruption of the mouse ERα gene using the Cre-loxP
recombination system. (A) Components of the ERα exon 3 wild-type allele, the targeted
allele following homologous recombination in ES cells, the floxed allele following deletion of the neomycin resistance gene (pGK-Neo) in ES cells, and the deleted ERα allele in Cre-recombinase transgenic mice. Abbreviations: B, BamHI; N, NheI; E,
Eco47III; H, HpaI; P, PmlI. (B) Genotyping PCR of tail clips of MMTV-Cre / ERαfl/fl
mice (lane 1), virgin ERαfl/fl (lane 2), and WAP-Cre / ERαfl/fl mice (lane 3). Lane 4
contains extract of a mammary gland from a parous WAP-Cre / ERαfl/fl mouse. The
MMTV promoter is active in skin, explaining the presence of the KO allele in the tail clip
in lane 1, whereas WAP is specific to the mammary gland during late pregnancy,
accounting for the 0.60 kb knockout allele in lane 4, but not lane 3.
Fig 2. ERα is required for mammary gland development. Whole mounts of mammary
glands (A-D) and H&E staining (E & F) from mature virgin MMTV-ERKO (A, C & E)
and ERαfl/fl mice (B, D & F). In ERαfl/fl, but not MMTV-ERKO mice, the #4 abdominal
mammary fatpad was fully occupied by the developed epithelial network.
Fig. 3. Dilation and inadequate branching of ducts in WAP-ERKO mice during 2nd
lactation. Whole mounts (A, C) and H&E (B, D) staining were used to analyze the
morphology of the mammary glands at second lactation day 1 of the WAP-ERKO
females (C,D) and ERαfl/fl controls (A,B). Note different magnifications in B and D,
emphasizing grossly enlarged ducts in mutant mice.
105 Fig. 4. Growth retardation in WAP-ERKO pups from the 2nd litter is attributable to
mother’s genotype. (A) WAP-Cre / ERαfl/fl pups nursed by their WAP-ERKO mothers
(WAP fl/fl-WAP fl/fl, n=5) were significantly smaller than control ERαfl/fl pups nursed by ERαfl/fl mothers (fl/fl-fl/fl). Beginning on the 4th day of lactation, a subset of WAP-
Cre / ERαfl/fl pups were fostered with lactating ERαfl/fl mothers (WAP fl/fl-fl/fl). After 4
days of fostering by an ERαfl/fl dam (lactation day 8), there was no longer a statistically
significant difference between WAP-Cre / ERαfl/fl and ERαfl/fl pup mass (n=4). Data are
presented as the mean ± 1 standard deviation. (B) Pairs of representative pups nursed for
10 days by birth mother or for 4 days of birth mother followed by 6 days of nursing by
foster dam as in (A).
Fig. 5. Loss of ERα and PR immunohistochemical staining in WAP-ERKO (WAP fl/fl),
but not ERαfl/fl (fl/fl) mammary glands of the second lactation, day 1. Nuclei of multiple
luminal epithelial cells in control mammary glands were positive for ERα and PR (C and
D), which were absent in the enlarged ducts of WAP-ERKO mice (A and B).
106 Supporting Information
Supporting Fig. 6. Introduction of loxP sites into the ERα locus. (A) The 4.6 kb BamHI- digested fragment of the targeted allele was detected with the 5’ probe indicated in Figure
1A by Southern blot in the ES cells (ES) and heterozygous floxed ERα mice (fl/+), but
not wild-type mice (+/+). (B) The 3’ probe detected a 4.0 kb BamHI fragment in ES cells
carrying the targeted allele (ES), and a 4.6 kb fragment following deletion of the floxed
neomycin resistance gene in heterozygous floxed ERα mice (fl/+). Only the 9.2 kb
fragment of the wild-type allele was detected in wild-type mice (+/+), which was also
detected in heterozygous mice and ES cells in both (A) and (B). (C) Homozygous
ERαfl/fl (fl/fl) and heterozygous ERαfl/+ (fl/+) mice were identified by genotyping PCR of
tail clips and detection of the 1.20 kb wild-type allele or the 1.28 kb floxed ERα allele
(see Figure 1A).
Supporting Fig. 7. Abnormal lubuloalveolar development and ductal branching during
first lactation cycle in WAP-ERKO mice. Whole mount of mammary glands from nursing WAP-ERKO (A) or control ERαfl/fl (B) female mice. Note “ballooning” acinus
in WAP-ERKO mammary gland (arrow).
Supporting Fig. 8. Detection of why acidic proteins in WAP-ERKO and control mice.
SDS-polyacrylamide gel electrophoresis and Coomassie blue staining of whey acidic
proteins (WAP) in the milk of lactating ERαfl/fl (fl/fl, n=2) and WAP-ERKO (WAP-fl/fl,
n=3) females. Note that the concentration of WAP is in the normal range in lactating
WAP-ERKO females. The first lane on the left contains the molecular weight marker.
Supporting Fig. 9. ER and PR expression in virgin WAP-Cre / ERαfl/fl mammary gland.
ERα (A) and PR (B) immunohistochemical staining in virgin WAP-Cre / ERαfl/fl
107 mammary gland, demonstrating that deletion of ERα and loss of its downstream target
PR is dependent on pregnancy and lactation. (C&D) Negative control staining without primary anti-ERα and -PR antibodies, respectively.
Supporting Fig. 10. Normal ER expression in mammary stromal cells and uteri of WAP-
ERKO females. Immunostaining was performed on the mammary and uterus tissue from
2nd lactation day 1 WAP-ERKO female to determine ERα expression. A) ERα staining in the mammary stroma; B) No primary antibody control in the mammary stroma; C) ERα staining in the uterus; D) No primary antibody control in the uterus.
108
Chapter IV
Mechanism of ERα-dependent developmental changes
in mammary gland
109
Abstract
The pregnancy-triggered remodeling in the mammary gland cycle is related to short-term
breast cancer risk and tumor invasion. Our results, from ER mammary epithelial
conditional knockout mice at embryonic and lactation stages, showed that epithelial ER is essential for luminal epithelial proliferation, differentiation and survival during
pregnancy and lactation stages. The phenotype includes the loss of side-branching as well
as alveolar development. To understand the mechanism of ER dependent developmental
changes in the mammary gland, mammary epithelial differentiation and proliferation
were analyzed. During the second lactation and third pregnancy of the WAP-ERKO
females, massive loss of luminal epithelial cells were observed, which was confirmed by
immunostaining of luminal and myoepithelial markers CK18, CK14 and CK5. The
detached epithelial cells in the lumens of dilated ducts further suggested that the loss of
ER compromised mammary epithelial proliferation and differentiation. These
observations support our conclusion that ER is essential for luminal epithelial
proliferation and differentiation during mammary gland morphogenesis.
110
Introduction
Alveolar morphogenesis
Upon pregnancy, the mammary gland undergoes massive tissue remodeling, represented by the first phase (epithelial proliferation) and the second phase (epithelial differentiation)
(Brisken, 2002; Richert et al., 2000). In response to hormones, including E2, P and PRL, the side-branches and alveolar buds begin to form and expand until clusters of alveoli fill the inter-ductal spaces. This developmental process, known as alveolar morphogenesis will prepare milk for the sucking pups upon parturition (Chapter I Figure 4)(Oakes et al.,
2006).
Mammary epithelial cells
The normal mammary epithelium is arranged as a bilayer including a luminal layer of secretory epithelial cells and a basal layer of myoepithelial cells. Expressing luminal epithelial markers cytokeratin 18 (CK18) and MUC1, the luminal epithelial cells are column-shaped and secrete milk during lactation (Adriance et al., 2005)(Fig. 1). Since most breast cancers are derived from ERα and PR-positive luminal epithelial cells, these tumor progenitors have received the most attention in breast cancer research. Spindle shaped myoepithelial cells lying underneath the luminal epithelium, are hybrids of muscle (‘myo’) and epithelial cells, expressing smooth muscle actin and myosin. In addition, they express specific markers including smooth muscle antigen (SMA) and cytokeratin 14 (Gudjonsson et al., 2002a) (Fig. 1). Accumulated evidence suggests that myoepithelial cells are natural tumor suppressor cells. Only a small percentage of breast cancers are derived from myoepithelial cells. They express multiple tumor suppressor
111 genes (such as Maspin, Cytokeratin-5, SMA and TIMP-1), which might be important for
preventing tumor cells in DCIS from invading the stroma (Faraldo et al., 2005; Polyak
and Hu, 2005).
An in-depth understanding of epithelial differentiation during alveolar morphogenesis continues to challenge the investigators. Elegant studies by Shackleton et al. (2006) and
Stingl et al. (2006) revealed that both luminal and myoepithelial cells are derived from the same mammary stem cells. A lone mammary stem cell can repopulate the whole mammary ductal network. Expressing unique cell surface markers such as CD24, CD29 and CD49, the stem cells may reside in the basal epithelial compartment. These results strengthen the emerging idea of breast cancer stem cells (Al-Hajj et al. 2003), an area of research, which holds great promise for developing novel therapies. Remarkably, the mammary tumor stem cells also express cell surface marker CD24, indicating their possible linage to mammary stem cells (Balic et al., 2006; Liu et al. 2006).
Signaling pathways in alveolar morphogenesis
The signaling pathways involved in alveolar morphogenesis have been reviewed in detail
by Hennighausen & Robinson (2005) and Oakes et al. (2006). The formation of milk
secreting units during pregnancy is synergistically regulated by P and PRL signaling. E2
acts in synergy with P to promote epithelial proliferation (Said et al., 1997). Progesterone
signaling is mediated by PR , including its downstream paracrine factors Wnt4 and
receptor activator of nuclear factor (NF)-κB ligand (RankL) in epithelial compartment
(Brisken et al., 2000; Brisken et al., 1998; Fata et al., 2000; Srivastava et al., 2003). Data
from the knockout animal models indicated that PR, RanKL and its ligand Rank are
112 required for alveolar morphogenesis. It is worth noting that RankL, working through
IKKα, activates Cyclin-D1 during lactogenesis (Oakes et al., 2006).
Prolactin receptor (PRLR) mediates PRL signaling. Stimuli from membrane PRLR and
EGF receptors are channeled to downstream genes via transcription factor Stat5 (Cui et
al., 2004; Long et al., 2003). Stat5 is phosphorylated by Jak2 and translocated to the nucleus to activate the downstream cascades of genes upon PRL induced PRLR
dimerization (DaSilva et al., 1996; Goffin et al., 2002; Hennighausen and Robinson,
2005). Stat5 activates transcription of several master regulators of alveolar
morphogenesis, including SOCS3, RankL, Cyclin D1 and Elf5, as well as the milk
proteins (Hennighausen and Robinson, 2005; Srivastava et al., 2003). Elf5, a major
downstream target of PRLR and Stat5, is a master regulator of the signaling cascade
controlling alveolar morphogenesis (Harris et al., 2006; Oakes et al., 2006; Zhou et al.,
2005). Elf5 knockout mice resembled mammary phenotypes of PRLR knockout mice.
Side-branching and alveolar-genesis were compromised in both heterozygous PRLR and
Elf5 knockout mammary glands. Furthermore, retroviral expression of Elf5 in PRLR-/-
mammary epithelial cells restored normal alveolar morphogenesis. These findings point
toward a common signaling pathway between PRL and Elf5.
ERα signaling in epithelial proliferation and differentiation
ERα has been established as a proliferation factor in cancer progression and mammary
gland development (Couse and Korach, 1999; Pearce and Jordan, 2004). Recently, Clarke
et al. (2005) showed that mammary stem cells might be ER positive. Consistent with this,
other groups showed that breast cancer stem cells were only found in ER positive
populations (Ponti et al., 2005). Furthermore, in vivo deregulation of ER signaling
113 profoundly affects lactogenesis, suggesting that ER is required for epithelial proliferation and differentiation. Repressor of ER Activity (REA) is an ER specific corepressor, which inhibits E2-induced ER transcriptional activity. ER activity is enhanced in REA knockout mice which caused a dramatic increase in alveolar development (Mussi et al., 2006).
Remarkably, several major ER downstream targets promote proliferation and are indispensable for alveolar morphogenesis, including cyclin D1, PR and IGF pathways
(Ismail et al., 2003; Planas-Silva et al., 2001; Surmacz and Bartucci, 2004). Epithelial cells cultured in vitro will die by apoptosis due to the loss of ERα and IGF signaling. On the other hand, cyclin D1 and IGF pathways have positive feedback on ERα transcriptional activity in mammary gland development.
Overall, epithelial ERα may regulate proliferation and differentiation of the epithelial cells. We hypothesize that ER in the epithelial compartment is indispensable for epithelial proliferation and differentiation in pregnancy and lactation stages.
In chapter II, we characterized MMTV-ERKO and WAP-ERKO conditional knockout mice. Our results showed that loss of ER and downstream PR led to abnormal mammary gland development in pregnancy and lactation stages, thereby compromising lactation in
WAP-ERKO mice. To understand the mechanism responsible for this mammary phenotype, we explored the role of ER in epithelial proliferation, differentiation and survival.
114
Results
Mammary gland development is compromised in pregnancy and lactation stages in
MMTV-ERKO females
Cre recombinase in the D line of the MMTV-Cre transgenic mice (Wagner et al. 2001)
used in this and other studies, is expressed in a spectrum of developmental stages ranging
from early embryonic to puberty. This temporal variation gave rise to heterogeneous
phenotypes in MMTV-ERKO mice. When Cre was expressed earlier, the mammary
gland development arrested at the neonatal stage and the MMTV-ERKO females were
infertile, generating a phenotype similar to genetic ERKO mice. When Cre was expressed in a later developmental stage the MMTV-ERKO females were fertile, indicating their hormone levels were normal and ER expression in the reproductive system might also be in normal range. However, the pups died in 48 hours. The MMTV-ERKO could not sustain the growth of swapped pups from control females, implying its mammary gland development is abnormal. When we analyzed the morphology of the lactating mammary gland on lactation day 6, we were surprised to observe remarkable similarity between the
MMTV-ERKO mammary glands and the WAP-ERKO mammary glands. Compared to the clusters of mature lobular alveoli surrounding the ducts in the control (fl/fl) mice very few mature lobular alveoli were present around the sparse mammary ducts in MMTV-
ERKO mammary gland (figure 2 A and B). Notably, some of the ducts were significantly dilated (Figure 2 A), indicating that the enlargement of the mammary ducts was due to failure of side-branching and alveolar development, which is different from the ductal dilation caused by senescence (aging). Similar to the enlarged alveoli in WAP-ERKO
115 females, a few enlarged alveoli were observed in the 4th mammary gland of the MMTV-
ERKO female, suggesting that ER is not only required for side-branching but also for alveolar formation (Fig. 3). Thus, the similarity between the mammary gland phenotype of MMTV-ERKO and WAP-ERKO females during lactation provide support to our hypothesis that ER is indispensable for alveolar morphogenesis.
Abnormal epithelial cells in WAP-ERKO mammary ducts during second lactation and third pregnancy. After analyzing the mammary phenotypes in both WAP-ERKO and MMTV-ERKO mice, we set out to explore the mechanism of the morphological changes in WAP-ERKO mammary glands. Normal mammary ducts are comprised of a bilayer of epithelial cells, the cuboidal luminal epithelia and the basal myoepithelia. The myoepithelia are characterized by the expression of smooth muscle actin (SMA), cytokeratin 5 (CK5) and 14 (CK14). MUC1, ERα, PR, cytokeratin 8 and 18(CK18) are specifically expressed in differentiated luminal epithelia (Adriance et al., 2005;
Gudjonsson et al., 2002b) (Fig. 1). We suspected that Loss of ERα and PR in dilated ducts may affect luminal epithelial proliferation and/or differentiation. Thereby, the morphology of the epithelial cells in WAP-ERKO females might be different from the controls. On the second lactation day 1 and day 4, the “luminal” epithelia in the dilated ducts of WAP-ERKO mammary glands are morphologically distinct from those in normal mammary ducts. They were small, flat and spindle-shaped which is similar to myoepithelial cells rather than normal luminal epithelial cells (Chapter II Figure 3).
Further analysis of the WAP-ERKO mammary gland in third pregnancy revealed similar ductal dilation and abnormal epithelial cells. Interestingly, the dilated ducts with
116 abnormal epithelial cells were correlated with successive breeding and were not common
in females that underwent only one cycle of pregnancy and lactation.
Normal expression of luminal and myoepithelial markers in WAP-fl/fl virgin mice.
To determine the identity of the epithelia in WAP-fl/fl females, immunostaining was
performed with antibodies against the basal and luminal markers. Immunostaining of
myoepithelial marker CK14 clearly distinguished the CK14 positive basal myoepithelial
cells from the CK14 negative column-like luminal epithelial cells in 2-month-old virgin
ERαfl/fl females. The CK14 expression pattern in age matched WAP-ERαfl/fl females was
similar to control females (Figure 4 and data not shown). As we expected, the cuboidal shaped luminal epithelial cells are CK18 positive. In addition, ER and PR expression in
WAP-fl/fl virgins are also similar to the controls. Therefore, the epithelial cells are
normal in the WAP fl/fl virgins (Figure 4).
Loss of luminal epithelia in the dilated ducts of WAP-ERKO females
The cellular specification and differentiation of mammary epithelium were further
analyzed in WAP-ERKO and control females during second lactation. The mammary
ducts in control fl/fl females showed CK14 positive myoepithelial cells and CK18
negative luminal epithelia (Fig. 5 C and D). In comparison with the controls, the thick
layer of column-shaped luminal epithelia was not detectable in WAP-ERKO mammary
gland, leaving the narrow ring of CK14 positive and CK18 negative myoepithelia at the
dilated ducts (Figure 5 A and B). Notably, the CK18 positive luminal epithelial cells and
CK14 positive myoepithelial cells were detected in a lobular alveolus beside the dilated
117 duct, indicating normal luminal epithelial cells were not present in the dilated ducts
(Figure 5 A and B red arrows). Loss of luminal epithelial cells was also observed in
WAP-ERαfl/fl females during first involution and 3rd pregnancy.
To further confirm our observation, we tracked the expression of another myoepithelial
marker and tumor suppressor, cytokeratin 5 (CK5) (Adriance et al., 2005).
Immunostaining of CK5 showed similar result as the CK14 staining. Very fewer CK5
negative epithelial cells were detected in the dilated ducts of WAP-ERKO females. Like the myoepithelial cells, the CK5 negative cells, which were flat and spindle-shape, were morphologically distinct from normal luminal cells (Fig. 6 B and B’). We counted the
CK5 positive and negative cells in 2 WAP-ERKO and 2 control females. The ratio of
CK5 positive cells to CK5 negative cells is 10:16 in control mice, but the ratio dropped to
10:6 in the KO females (Figure 6 C). Overall, we conclude that loss of ERα expression compromised luminal epithelial proliferation, differentiation and survival.
Apoptotic epithelial cells in the lumen of mammary ducts
ER is a proliferation factor in mammary gland development and breast cancer cells.
Therefore, loss of ER may affect epithelial proliferation and survival. To confirm that loss of luminal epithelial cells indeed represented lack of cell proliferation, we analyzed
PCNA staining in the 2nd lactation day 1 mammary glands. WAP-ERKO mammary glands showed a significant decrease of PCNA positive luminal cells compared to ERfl/fl
controls, suggesting that the loss of ER arrested cell cycle progression (data not shown).
Transcription factor Gata-3 is required for luminal epithelial differentiation. Gata-3 may
regulate ER expression in luminal epithelial cells (Asselin-Labat et al., 2007; Kouros-
Mehr et al., 2006). The Gata-3 null luminal cells were ER negative and detached from
118 mammary ducts. We suspected that luminal epithelial cells might lose attachment to the
mammary duct in WAP-ERKO mice. As we expected, numerous cells were detected in
MMTV-ERKO mammary gland during lactation. Macrophages, monocytes and engulfed
apoptotic cells were also observed. In WAP-ERKO mammary gland during 2nd lactation,
epithelial cells were detected in the ductal lumen as well. However, few epithelial cells
were detected in the control glands indicating that ERα is required for luminal epithelial
differentiation and proliferation (Figure 7).
Discussion
Ductal dilation and abnormal alveologenesis
Ductal dilation and abnormal lactogenesis were commonly observed in WAP-ERKO mammary glands during and after the second lactation. Though the abnormal alveoli
were observed as early as on first lactation day 4, the mammary gland function is largely
normal until the second lactation. The whole mounts staining of WAP-ERKO mammary
gland provided clues for the development of the ductal dilation. In the WAP-ERKO
mammary glands, the mammary ducts were not universally dilated and some balloon
shaped side-branches that bore no acini and lobular alveoli were observed. Therefore,
loss of ERα may have compromised the normal development of committed side-branches.
Normally, epithelial proliferation is followed by epithelial differentiation in lactogenesis,
suggesting that epithelial proliferation might be required for epithelial differentiation.
However, the epithelial cells in WAP-ERKO mammary gland may still proliferate but
cannot further branch and differentiate into lobular alveoli. Therefore, the ductal dilation
in WAP-ERKO mammary gland represents a secondary effect of compromised side-
119 branching and alveolar development. Our suspicion is further supported by the loss of
luminal epithelial cells in the abnormal mammary ducts in WAP-ERKO mammary
glands. Thus, we conclude that ER in epithelial compartment is a master regulator of
alveolar morphogenesis. Interestingly, PRLKO, PRLRKO, PRKO and AIB1 transgenic
mice all showed ductal dilation after hormone stimulation (Nelson Horseman personal
communication) (Long et al., 2003; Lydon et al., 1999; Torres-Arzayus et al., 2004).
Presumably, ER expression in the luminal epithelial cells is maintained in these mice and
the luminal cells are morphologically normal, suggesting the loss of luminal cells is not
due to the loss of PR and PRLR.
The severe phenotypes in WAP-ERKO females are correlated with successive pregnancy
and lactation. While the phenotypes are less obvious in non-successive pregnant and
lactating females, suggesting there is a compensation for the loss of ERα positive epithelial cells. In addition, the phenotype is more obvious in older WAP-ERKO females
(pregnant after 3 month-olds) than the younger ones (pregnant before 2 months of age).
This may reflect the decreased activity of mammary stem cells in the aging process
(Russo et al., 2005). Since ER is mainly knocked out in differentiated WAP-positive luminal epithelial cells (Wagner et al., 2002; Wagner et al., 1997), the phenotype may not be due to the ER knockout in luminal progenitors and epithelial stem cells. However, overlapping of pregnancy and lactation at the same time limited the supply of ER protein
(via ER deletion) and accelerated ERα degradation in luminal epithelial cells (Shao et al.,
2004), thereby causing the abnormal side-branching and alveolar development. Overall,
120 the mammary phenotype of WAP-ERKO females is a combined result of stem cell
depletion and ERα protein exhaustion.
Epithelial differentiation
We showed here for the first time that ERα is a critical regulator of luminal epithelial
survival, proliferation and differentiation. This finding adds new in vivo evidence for ER
in cell-fate decisions in mammary gland development. Previous research indicated that
Gata-3 is critical for luminal epithelial development and ERα might be downstream of
Gata-3. The Gata-3 negative luminal epithelial cells detached from mammary ducts, but
the apoptosis is a secondary effect in the Gata-3 null epithelial cells (Kouros-Mehr et al.,
2006). Gata-3 inhibits epithelial proliferation and promotes epithelial differentiation.
The mammary phenotypes in WAP-ERKO females are not identical to Gata-3
conditional knockout mice. Luminal epithelial proliferation was decreased in WAP-
ERKO females, while there was an increase of luminal epithelial proliferation in Gata-3
null epithelial cells. The loss of luminal epithelial cells might be due to the activation of
the apoptosis pathways in the absence of ER. These results reflect the distinct roles of
Gata-3 and ER in mammary gland development. ER is critical for the survival and
proliferation of luminal epithelial cells and sustaining epithelial differentiation by maintaining PR expression. Gata-3 is an epithelial differentiation factor.
ER has been established as a proliferation factor in the presence of high levels of
circulating E2 in mammary gland. Though ER downstream target PR is required for
epithelial differentiation, whether ER is directly involved in epithelial differentiation is
121 not known. Pechoux et al. showed that luminal epithelial cells can give rise to myoepithelia and not vise versa (Pechoux C et al. 1999). When luminal epithelia were cultured in medium supporting myoepithelial differentiation, they gradually lost expression of CK18 and CK19 and gained expression of α-SMA, vimentin and CALLA.
It is still not clear whether mammary stem cells are ER positive. Some results showed that some mammary stem cells might be ER positive (Booth and Smith, 2006; Clarke et al., 2005); Others showed that ER positive status is not related to mammary stem cells
(Asselin-Labat et al., 2006; Sleeman et al., 2007). We cannot rule out that ER status affects epithelial differentiation; therefore some luminal epithelial cells may gain
characteristics of myoepithelial cells in the absence of ER.
The hint of loss of luminal epithelial cells on tumorigenesis
Luminal epithelial cells are the precursors of most breast cancer cells. Loss of
myoepithelial function is almost universally associated with the development of breast
cancer (Polyak and Hu, 2005). We observed massive loss of luminal epithelial cells in
mammary ducts during second lactation and third pregnancy in WAP-ERKO mice. The
ratio of CK5 cells to CK5 negative cells increased from 10: 16 to 10: 6 in the WAP-
ERKO mammary ducts. CK5 is a tumor suppressor expressed in the natural tumor
suppressor, myoepithelial cells. Loss of tumor suppressor expression and/or loss of
myoepithelial cells correlated with breast cancers (Adriance et al., 2005; Barsky and
Karlin, 2005). Higher ratio of CK5 positive cells to CK5 negative cells in WAP-ERKO
suggested that there might be lower occurrence rate of ER positive breast cancer and
overall breast cancer rate in these mice. Though ERα might not be an oncogene, it might
122 be required for tumor initiation, providing the nursery for development of luminal derived breast cancers. Thereby, loss of ER may prevent the occurrence of breast cancer.
No obvious mammary phenotype in first involution in WAP-ERKO mammary
glands
Mammary phenotypes in second lactation in WAP-ERKO might be due to early
activation of involution. The morphology of WAP-ERKO mammary gland in 2nd
lactation is similar to the mammary gland during involution, including massive loss of
luminal epithelial cells, presence of apoptotic cells in the ductal lumen and increased
number of adipocytes. However, we did not detect dramatic difference between the
WAP-ERKO and control glands in forced wean during first lactation. Incomplete ER
knockout by WAP-Cre might be the reason. Both REA knockout mice and AIB1
transgenic mice showed delayed involution, suggesting ER conditional overexpression
could be an alternative approach to address the role of ER in involution. We predict that
tetracycline-controlled ER overexpression in mammary gland will delay the involution
process.
123 Materials and Methods:
Whole-mounts staining
Whole-mounts staining was performed as previously described (Mueller et al., 2002).
Mice were killed in CO2 tank and inguinal mammary fat pads (the forth mammary gland)
were excised and fixed for a minimum of overnight in Carnoy’s solution (60% ethanol,
30% chloroform, and 10% glacial acetic acid). The fixed glands were washed in 70%
ethanol for 15 min and then rinsed in water for 5 min. The mammary glands were stained
overnight at 4°C in carmine alum stain (1 g carmine and 2.5 g aluminum potassium
sulfate in 500 ml water). The glands were then dehydrated progressively in 70%-95%-
100% ethanol, cleared in xylene for 2 h, and mounted on glass slides with Permount
(Fisher Scientific, Suwanee, GA). Mammary whole mounts were photographed using a
Nikon Microphot – FXA microscope and SPOT 4.0.9 for Windows (Contact Diagnostic
Instruments, Sterling Heights, MI).
Hematoxylin & eosin (H&E) staining
Mammary glands were fixed in 10% neutral buffered formalin for 48h in room
temperature. Then the tissues were placed in 70% ethanol, dehydrated and paraffin- embedded. For H&E staining, the tissue section (5 µm) were: 1. cleared by 2 changes of xylene, 10 minutes each; 2. Re-hydrated in 2 changes of absolute alcohol, 5 minutes each;
3. 95% alcohol for 2 minutes and 70% alcohol for 2 minutes. 4. Washed briefly in distilled water. 5. Stained in Harris hematoxylin solution for 8 minutes. 6. Washed in running tap water for 5 minutes. 7. Differentiated in 1% acid alcohol for 30 seconds. 8.
Wash in running tap water for 1 minute. 9. Bluing in 0.2% ammonia water or saturated
lithium carbonate solution for 30 seconds to 1 minute. 10. Wash in running tap water for
124 5 minutes. 11. Rinse in 95% alcohol, 10 dips. 12. Counterstained in eosin-phloxine B solution (or eosin Y solution) for 30 seconds to 1 minute. 13. Dehydrate through 95% alcohol, 2 changes of absolute alcohol, 5 minutes each. 14. Clear in 2 changes of xylene,
5 minutes each. 15. Mount with xylene based mounting medium. Digital photomicrographs were acquired with a Nikon Microphot – FXA microscope and SPOT
4.0.9 for Windows (Contact Diagnostic Instruments, Sterling Heights, MI).
Immunohistochemistry. Slides were boiled in 1X citrate buffer (pH 6.0) for 20 minutes
for antigen retrieval. K18 (1:30, cat. #: 61028, Progen) IgG were used with the Histo-
mouse-MAX kit (Zymed Lab cat. #: 87-9551) to detect K18 expression according to the
manufacturer’s instructions. Immunostaining with CK14 (1:1000, PRB-155P,
COVANCE) and CK5 (1:1000, PRB-160P COVANCE) antibodies were performed
according to the manufacturer’s instructions. Biotinylated secondary antibody (goat anti-
rabbit IgG) and the avidin-biotin blocking kit were from Invitrogen (cat. #s 50-235Z and
00-4303) and Vectastain ABC kit was from Vector Lab. Digital photomicrographs were
acquired with a Nikon Microphot – FXA microscope and SPOT 4.0.9 for Windows
(Contact Diagnostic Instruments, Sterling Heights, MI).
125
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Srivastava, S., Matsuda, M., Hou, Z., Bailey, J.P., Kitazawa, R., Herbst, M.P., and Horseman, N.D. (2003). Receptor activator of NF-kappaB ligand induction via Jak2 and Stat5a in mammary epithelial cells. The Journal of biological chemistry 278, 46171- 46178.
Surmacz, E., and Bartucci, M. (2004). Role of estrogen receptor alpha in modulating IGF-I receptor signaling and function in breast cancer. J Exp Clin Cancer Res 23, 385- 394.
Torres-Arzayus, M.I., Font de Mora, J., Yuan, J., Vazquez, F., Bronson, R., Rue, M., Sellers, W.R., and Brown, M. (2004). High tumor incidence and activation of the PI3K/AKT pathway in transgenic mice define AIB1 as an oncogene. Cancer Cell 6, 263- 274.
Wagner, K.U., Boulanger, C.A., Henry, M.D., Sgagias, M., Hennighausen, L., and Smith, G.H. (2002). An adjunct mammary epithelial cell population in parous females: its role in functional adaptation and tissue renewal. Development 129, 1377-1386.
Wagner, K.U., Wall, R.J., St-Onge, L., Gruss, P., Wynshaw-Boris, A., Garrett, L., Li, M., Furth, P.A., and Hennighausen, L. (1997). Cre-mediated gene deletion in the mammary gland. Nucleic Acids Res 25, 4323-4330.
Zhou, J., Chehab, R., Tkalcevic, J., Naylor, M.J., Harris, J., Wilson, T.J., Tsao, S., Tellis, I., Zavarsek, S., Xu, D., et al. (2005). Elf5 is essential for early embryogenesis and mammary gland development during pregnancy and lactation. The EMBO journal 24, 635-644.
129
Figures:
Figure 1
Mammary ductal structure and mammary epithelial cells
Mammary duct }
Stem cells/progenitors Stroma Stromal cells } Alveolar cells (ER+) acinus Myoepithelial cells (CK14)
Luminal epithelial cells (ER+, PR+, CK18)
130
Figure 2
Wholemounts H&E A B MMTV-ERKO
C D Control
131
Figure 3 Wholemounts H&E A B MMTV-ERKO
CD Control
132
Figure 4
133
Figure 5
134
Figure 6
AB
WAP fl/fl fl/fl C B’
140
120
100
80 Basal epithelial cells 60 Luminal epithelial cells 40
20
0 fl/fl WAP fl/fl WAP fl/fl
135
Figure 7
136
Figure legends
Figure 1. Mammary ductal structure and mammary epithelial cells. Mammary glands in pregnancy and lactation stages are comprised with three types of epithelial cells, the column-shaped (cuboidal) luminal epithelial cells (shown in blue); spindle-shaped Myoepithelial cells (yellow) and alveolar cells (green). The mammary stroma, which contains stromal cells (dark green), is outside of myoepithelial cells.
Figure 2. Ductal dilation and lack of side-branching in MMTV-ERKO females during lactation. Whole mounts (A and C) and H&E (B and D) staining was performed to determine the morphology of MMTV-ERKO(A and B) and the control mammary gland(C and D). A&C Scale bar = 100 μm; B&D Scale bar = 50 μm.
Figure 3. Abnormal alveoli in MMTV-ERKO mice during lactation. Whole mounts (A and C) and H&E (B and D) staining was performed to determine the morphology of MMTV-ERKO(A and B) and the control mammary gland(C and D). A&C Scale bar = 100 μm; B&D Scale bar = 50 μm.
Figure 4. Normal K14 and K18 expression in WAP-fl/fl virgins. Immunohistochemical analysis was performed on tissue sections of paraffin-embedded mammary glands from two-month-old WAP-ERαfl/fl virgins. The sections were stained with anti-CK14 (A) and anti-CK18 (C) antibodies, developed with Vectastain ABC kit, and counterstained with hematoxylin. No primary antibody controls were shown in B and D. Myoepithelial cells are K14 positive and K18 negative. Luminal epithelial cells are K18 positive and K14 negative. Scale bar = 50 μm.
Figure 5. Loss of CK14 negative luminal cells in WAP-ERKO females. Immunohistochemical analysis was performed on serial sections of paraffin-embedded mammary glands from WAP-ERKO (A and B) and control females (C and D) on 2nd lactation day 1. The sections were stained with anti-CK14 and anti-CK18 antibodies, developed with Vectastain ABC kit, and counterstained with hematoxylin. Myoepithelial cells are K14 positive and K18 negative. Luminal epithelial cells are K18 positive and K14 negative. Scale bar = 25 μm.
Figure 6. Loss of K5 negative cells in WAP-ERKO females. Immunohistochemical analysis was performed on serial sections of paraffin-embedded mammary glands from WAP-ERKO (B and B’) and control females (A) on 2nd lactation day 1. The sections were stained with anti-CK5 antibodies, developed with Vectastain ABC kit, and counterstained with hematoxylin. The ratio of Basal and luminal cells is changed in KO mammary gland. Basal cells are K5 positive. Basal (K5+):Luminal(K5-)=10:14 in fl/fl mice; Basal(K5+):Luminal(K5-)=10:6 in WAP-ERKO. Scale bar = 100 μm.
Figure 7. Apoptotic epithelial cells in the lumen of WAP-ERKO mammary ducts. Immunostaining was performed to determine the expression of CK5 in WAP-ERKO (A
137 and B) and fl/fl mammary gland(C). Apoptotic cells were indicated by red arrows. Scale bar in A and C are 50 nm; 25 nm in B.
138 Chapter V
Summary and discussion
Summary
ER was revealed as a major regulator of postnatal mammary gland development by
Korach and colleagues’ seminal studies employing ER genetic knockout mice. However,
ER regulates mammary gland development at the mammary gland and whole-organism levels by regulating the hormones. To dissect the role of ER in epithelial compartment, exon 3 of ER was deleted at different time points in mammary epithelial cells, utilizing
Cre-loxP-mediated conditional knockout system. The studies presented in this thesis established the role of ER in mammary gland development and epithelial survival during pregnancy and lactation stages. In chapter II, two ER mammary gland conditional knockout animal models were established. The results from both animal models
demonstrated that epithelial ERα is essential for side-branching and lobuloalveolar
development. Importantly, epithelial ER is indispensable for luminal epithelial
proliferation, differentiation and survival. In addition to normal mammary gland
development, ERα is a core regulator of breast cancer pathogenesis, and multiple signal
transduction pathways converge on ERα activity to promote breast tumor progression.
We found that NRCC-A, a novel co-regulator of ERα, not only enhances ERα
transcriptional activity but also promotes breast cancer cell proliferation.
Conclusion
Summary and the rationale of conditional ERKO
139 ER is a critical nuclear hormone receptor that mediates tissue specific biological
functions of E2 in different target tissues and organs, as demonstrated in genetic knockout mice (Couse and Korach, 1999; Pearce and Jordan, 2004). Mammary gland development was arrested in neonatal stage in ERKO mice, revealing the essential role of
ER in ductal elongation during puberty. However, ER determines mammary gland development at the local and whole-organism levels by regulating circulating pituitary
and ovarian hormones (Bocchinfuso et al. 2000). It is impossible to assess the
consequences of mammary-gland-specific ER inactivation on mammary gland development with a whole-body genetic knockout. To circumvent this difficulty and
explore whether ER exhibits unique functions at various stages of mammary gland
development, we deleted exon 3 of ER at different time points in mammary epithelium
by using Cre-loxP-mediated recombination. This study demonstrates that ER in epithelial
compartment is essential not only for pregnancy-mediated cell proliferation and
differentiation but also for the survival of mammary epithelium.
The mammary phenotypes in conditional ERKO mice
We successfully generated two mammary epithelial ER conditional knockout animal
models. Both WAP-ERKO and MMTV-ERKO show similar phenotypes during
pregnancy and lactation stages, represented by the compromised side-branching, lack of
alveolar morphogenesis, ductal dilation and the loss of luminal epithelial cells. It is the
first time that the in vivo function of ER in mammary gland cycle was revealed by the ER
conditional loss-of-function assay. In the WAP-ERKO mice, the severe phenotype in the
mammary gland was correlated with the loss of ER and its target PR.
140 Loss of side-branching and alveolar morphogenesis in second lactation
We observed loss of side-branching and lack of mature lobuloalveoli during second lactation and third pregnancy and established that epithelial ER is critical for normal lactation. This phenotype is correlated with the progressive loss of ER and its target PR after the second pregnancy. Abnormal mammary gland development was observed as early as first lactation day 4. However, lactation and mammary gland development were largely normal in the first lactation. A similar result was seen in Erbb4 conditional knockout mice (Long et al., 2003). There might be a selective pressure for the mammary epithelial cells to retain the expression of ER. On the other hand, incomplete ER knockout by WAP-Cre in first lactation might be another reason that the mammary gland is still functional during the first lactation.
ER signaling in mammary gland development
It is interesting that knockout of several key regulators in alveolar morphogenesis, including PR, Cyclin D1, RankL, PRLR, Elf5 and PRL, produced similar phenotypes as seen in WAP-ERKO females (Harris et al., 2006; Horseman et al., 1997; Lydon et al.,
1995; Oakes et al., 2006; Sicinski et al., 1995; Zhou et al., 2005)(Fig. 1). In these KO mice, the epithelial ductal network is largely normal until pregnancy. Epithelial transplantation assay showed that the knockout epithelial cells do not undergo further side-branching and can not form alveoli in the wild type fat pad, indicating epithelial cells are essential for alveolar morphogenesis. Upon hormone stimulation, though PRKO,
PRLKO and PRLRKO females had no alveolar development, they had similar ductal dilation as WAP-ERKO females (Fig. 1). Remarkably, these factors belong to either PR or PRLR pathways. Cyclin D1 and RankL are the downstream targets of PR; cyclin D1,
141 RankL and Elf5 are the downstream targets of PRL and its receptor PRLR. Furthermore,
PR and cyclin D1 are direct downstream targets of ER in mammary gland development.
E2 activates PR and cyclin D1 gene expression via ER (Mussi et al., 2006). The
expression of PRLR may also be activated by ER. In comparing with the members of PR
and PRLR pathways, ER is not only important for alveolar morphogenesis, but also
critical for ductal elongation during puberty. It is believed that ER is an upstream
regulator of PR and PRLR signaling in mammary gland development. Overall, ER
signaling in adult mammary gland development might be mediated by PR, PRLR and
cyclin D1 signaling. Thus, we speculate that loss of downstream PR, cyclin D1 and
PRLR secondary to loss of ER signaling in WAP-ERKO mice led to the abnormal mammary gland development during pregnancy and lactation stages.
AB
WAP-ERKO PrlKO+E2 & Prl (OVEX)
WT PRKO + Pituitary Isograft PrlKO (Dr. Horseman), PRKO (Dr. Lydon et al Cancer Research 1999)
Figure 1 Ductal dilation in PRKO, WAP-ERKO and PRLKO mammary glands. H&E staining on WAP-ERKO (A), Hematoxylin staining on PRLKO (B), BrdU staining on WT (C) and PRKO (D). B is kindly provided by Dr. Horseman. C and D are from Lydon et al., Cancer Research 1999.
142 ER in the epithelial compartment is critical for cell proliferation and differentiation
during pregnancy and lactation stages
There was very limited epithelial development in WAP-ERKO knockout mice in second
pregnancy and lactation. Several scenarios may explain these results. Epithelial
proliferation might be required for generating sufficient numbers of epithelial cells for
epithelial differentiation. ER is a potent proliferation factor in mammary gland
development, so it is not surprising that loss of ER compromised epithelial proliferation.
The proliferation effect of ER is further amplified by some downstream paracrine and autocrine factors. Therefore, the reduced proliferation is also related to the loss of downstream proliferation factors in luminal epithelial cells. Cyclin D1, which activates and provides substrate specificity of its partners CDK4 and 6, is a critical component of the core cell cycle machinery in eukaryotic cells (Sicinski et al., 1995). Cyclin D1 is a direct transcriptional target of ER in mammary gland development and an indirect downstream target of both PR and PRLR cascade (Mussi et al., 2006). There was no alveolar morphogenesis in cyclin D1 null mice (Sicinski et al., 1995). Thus, lack of proliferation in WAP-ERKO may due to the combined result of loss of ER and cyclin D1.
Both PR and PRLR pathways govern epithelial differentiation. Loss of either pathway
compromises side-branching and alveolar development. We showed that PR expression is
lost in WAP-ERKO mammary gland during second lactation, suggesting that ER
regulates epithelial differentiation via PR. Whether ER is required for maintaining
epithelial differentiation in the absence of PR remains to be addressed.
Luminal epithelial and myoepithelial cells are derived from same mammary stem cells
(Shackleton et al., 2006; Stingl et al., 2006). ER might be required for adult mammary
143 stem cells and progenitor cells (Clarke et al., 2005). Though normally WAP is expressed
in differentiated epithelial cells, a leaky expression of the WAP-Cre transgene may
knockout ER in mammary stem cells and epithelial cells, thereby blocking epithelial cell
differentiation. Alternatively, mammary stem cells and/or progenitor cells might be ER
negative but need paracrine signal from ER positive cells to proliferate and differentiate.
This scenario is supported by the finding that cyclin D1 is a direct target of ER and downstream PR and PRLR pathways.
ER as a survival factor of luminal epithelium
Mammary epithelial cells differentiate into alveolar epithelial cells, which are functional
throughout the lactation period, during the later stage of pregnancy. We observed the loss
of luminal epithelial cells during second lactation, represented by the loss of CK14
negative and CK18 positive cuboidal epithelial cells. Since ER downstream signaling are
known as survival factors in epithelial cells and breast cancer cells (Hadsell et al., 2002),
we suspect that epithelial cells detected in ductal lumen were undergoing apoptosis.
Previous studies have established that IGF-1 signaling might be a downstream survival
signal of ER. The expression of IGF-1, IGF-1 receptor (IGF-1R) and IRS-1 were
upregulated by ER (Surmacz and Bartucci, 2004). The loss of IGF-1 signaling leads to
epithelial apoptosis in primary culture. ER, PR and PRLR are co-expressed in the same
luminal epithelial cells (Grimm and Rosen, 2003, 2006). It is not surprise that the loss of
ER in WAP-ERKO will lead to the loss of PR and PRLR. Thereby, the loss of PRLR
signaling in WAP-ERKO mammary gland may induce the epithelial apoptosis and lack
of alveolar development. Prolactin and PRLR signaling was mediated by transcription
factor Stat5. Stat5 has been shown as a survival factor during alveolar morphogenesis
144 (Cui et al., 2004). Increased apoptosis was observed at pregnancy day 15 in Stat5 mammary specific conditional knockout mice (Cui et al., 2004). Thus, the loss of IGF-1,
PR and PRLR signaling in WAP-ERKO mammary gland compromised side-branching and alveolar development and may result in epithelial apoptosis (Fig. 2).
Normal Mammary ERKO mammary gland gland IGF-1 C PrlR C ERα PR CyclinD1
Luminal epithelial cells Apoptotic luminal epithelial cells
Myoepithelial cells Epithelial progenitor cells Mammary stroma
Figure 2 Epithelial ER and its targets are required for epithelial proliferation, differentiation and cell survival. The severe phenotype after the successive pregnancies is due to loss of luminal epithelial progenitors secondary to loss of ER signaling. ER deletion eliminated some epithelial progenitor cells that derived from the WAP positive epithelial cells in first lactation. In addition, high E2 and P levels during pregnancy not only promote ER deletion by WAP-Cre, but also enhanced ER and PR protein degradation. Furthermore, we cannot rule out the possibility that ER is required for maintaining the adult mammary stem cell niche. Therefore, luminal epithelial cells that cannot proliferate and differentiate in the absence of ER, may undergo massive apoptosis, resulting in the loss of luminal epithelial cells on the mammary duct. Thus, we concluded that ductal dilation, lack of tertiary branching and alveolar development in WAP-ERKO mice are due to the depletion of functional luminal epithelial cells and their progenitors.
ER and breast cancer
There are two possible roles of ER in breast cancer development: (1) ER sustains the growth of some breast cancers. The role of ER as a proliferation factor in ER positive cancer has been well documented and accepted (Fuqua, 2001; Grimm and Rosen, 2006;
Key, 1999). (2) ER is a breast cancer initiation factor. Our and others’ results suggest that
145 ER might be a required factor for luminal derived breast cancer initiation (Holst et al.,
2007). In the absence of ER, myoepithelial cells still retain expression of CK14 and CK5,
indicating their survival might not depend on ER. In contrast, luminal epithelial cells were not maintained in the absence of ER. Presumably, they died by apoptosis. Notably,
ER gene was frequently amplified in benign and precancerous breast diseases, suggesting the role of ER in breast cancer initiation. Significantly, 99% of breast cancers with ER
gene amplification are ER positive (Holst et al., 2007). Thus, ER might be an
indispensable survival signal for breast cancer initiation.
Discussion of NRCC:
NRCC-A and ER ligand-independent transcriptional activity
Like many other coactivators, NRCC-A enhances ER transcriptional activity in both
ligand dependent and ligand independent fashions. Consistent with this, NRCC-A
interacts with ER in the presence or absence of E2. It is interesting that the enhancement
is at the highest level after 12-16 hours of ligand treatment, relative to controls. In the
absence of ligand, ER interacts with NCoR corepressor complex on the silenced pS2
promoter. Ligand binding triggers the release of the NCoR complex and allows
interaction of ER with p160 family members, which will in turn recruit other coactivators
required for transcriptional activation. We suspect that the presence of NRCC-A may
destabilize the interaction between the NCoR complex and ER. On the other hand,
NRCC-A may facilitate p160 family members to interact with ER, thereby enhancing
ligand-independent transcriptional activity.
NRCC-A and ER ligand-dependent transcriptional activity
146 The mechanism by which NRCC-A affects ligand dependent transcriptional activity of
ER is not clear. Some ligand dependent corepressors, such as LCoR and REA, interact
with ER in the presence of E2. They recruit HDACs to the ER transcriptional activation complex and inhibit ligand dependent transcriptional activity. How this ligand dependent repression is overcome in vivo is not understood. Since NRCC-A has the effect of accelerating ligand-dependent transactivation of ER, it may have a role in counterbalancing the inhibitory effect of ligand dependent repression. It is not clear whether NRCC-A can interact with other ER coactivators such as CBP/p300 and the mediator complex. An alternative action of NRCC-A upon ligand binding would be facilitating the recruitment of other coactivators.
Which class of coactivator does NRCC belong to?
The presence of NR-boxes in the sequence and ligand-independent transcriptional
activity suggest that NRCC-A is not a secondary co-activtor and may represent a novel
type of hormone coactivator that is involved in corepressor and coactivator complex
exchange. The identified secondary coactivators do not enhance ER ligand-independent
transcriptional activity and may not interact with ER directly. They interact with the N
terminal bHLH-PAS domain of GRIP1.
NRCC in ER-dependent and independent cell proliferation
NRCC proteins, presumably NRCC-A, are important for E2 stimulated cell proliferation.
This proliferation effect is consistent with our observation that NRCC affects ER transcriptional activity. NRCC may be a member of the ER transcriptional complex, thereby promoting ER dependent cell proliferation. Another question relates to the molecular basis of the observed ER independent cell proliferation by NRCC-A. We have
147 considered two possibilities. (1) NRCC may promote cell proliferation through other
hormone receptors. For example, NRCC-A enhanced AR transcriptional activity in our
reporter assays. Though AR is not present in Hela cells, GR and PR are expressed. It will
be interesting to test whether this effect is GR or PR related. (2) Hormone receptor independent proliferation. Some ER coactivators are cell proliferation factors, such as
cyclin D1 (Zwijsen et al., 1998; Zwijsen et al., 1997). Therefore we cannot rule out that
the proliferative effect of NRCC is hormone receptor independent. Ligand independent-
activation of ER may relate to tamoxifen (TAM) resistance. AIB1, which enhanced
ligand-independent transcriptional activity, promoted cancer cell proliferation in the
presence of TAM. So, we will test the role of NRCC proteins in TAM resistance.
Coiled-coil domain and proteins interacting with NRCCs
NRCC-A is predicted to contain a coiled-coil domain, which is important for dimer and
trimer formation and protein-protein interaction. Many important transcription factors and co-regulators contain coiled-coil domains, such as the oncogene c-myc and corepressor GPS2. Like many other Coiled-coil domain proteins, NRCCs may also form homo- or hetero- dimers and may be a member of a protein complex in the nucleus.
Identification of NRCC contact proteins will help us to understand the role of NRCC in transcriptional regulation as well as cell proliferation.
Why are there so many coactivators?
The diverse physiological functions of ER and other NRs indicate that receptor specific and tissue specific factors define their roles. Furthermore, general transcription initiation and elongation is a fine-tuned dynamic process which needs collaboration of multiple factors to maintain cycling of the complex,(Lonard and O'Malley, 2006; Perissi and
148 Rosenfeld, 2005). The identified co-factor is only a small portion of the transcriptional
complex. Therefore, newly identified coactivators will continue to be a prime source for the discovery of new molecular events and mechanisms in signaling control and anti- estrogen treatment. For example, it is not known which factors cause the antagonist vs agonist action of tamoxifen in breast and uterus, respectively. We predict that identification of some tissue specific co-regulators may reveal the molecular mechanism for tamoxifen’s tumorigenic effect in uterus.
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152
Chapter VI
Ongoing/Future directions
A. The role of ERα in AIB1-induced tumors
Enhanced ER transcriptional activity and increased ER expression are major risk factors
in breast cancer (De Assis and Hilakivi-Clarke, 2006). Overexpression of coactivators
significantly enhances ER transcriptional activity. AIB1, also known as SRC3, RAC3 and
ACTR, is the major p160 family coactivator of ER in mammary gland. AIB1 gene was
cloned from a frequently amplified region of chromosome 20q11-13 in human breast and ovarian cancers (Anzick et al., 1997; Xu and Li, 2003). The AIB1 gene is amplified in breast cancers, hepatocellular carcinoma (HCC) and primary gastric cancers (Anzick et al., 1997; Sakakura et al., 2000; Wang et al., 2002). The mRNA of AIB1 is overexpressed in up to 60% of breast tumors. AIB1 knockout animals are dwarf and exhibit an impaired mammary ductal network, suggesting a critical role for AIB1 in mammary gland development (Xu et al., 2000). AIB1, unlike other members of the p160 family coactivators, stabilizes ER in the presence of E2 (Shao et al., 2004). The delayed v-Ha- ras-induced cancer initiation and progression seen in AIB1 knockout mice is consistent with a prominent role for this coactivator in breast cancer development (Kuang et al.,
2004).
AIB1 was recently established as an oncogene. Overexpression of AIB1 in mice leads to development of malignant mammary tumors (Torres-Arzayus et al., 2004). 76% of AIB1 transgenic mice developed various types of tumors. About 1/3 of the all tumors were mammary adenocarcinomas. Interestingly, the expression of ERα and its downstream
153 target IGF-1 were increased in AIB1 transgenic mice and the majority of breast tumors
were ER positive. Thus, we hypothesize that: breast tumorigenesis in AIB1 transgenic
mice is ER-dependent.
To address our hypothesis, ERα will be knocked out in the context of AIB1
overexpression in vivo and in vitro. In the in vivo knockout system, tetracycline-
controlled Cre recombinase will be introduced into AIB1/ERfl/fl mice to delete ER at
different time points to test its role in tumor initiation and tumor progression. At the same
time, ER positive and negative cancer cell lines will be established from tumors and their
cell linage will be analyzed. ER will be knocked out in vitro with Adenovirus expressing
Cre Recombinase or dox.
We predict that deletion of mammary epithelial ER in the AIB1 transgenic background
will attenuate initiation and progression of breast cancer. In contrast, hormone
stimulation (pregnancy or E2, P and PRL treatment) will accelerate the progress of
tumorigenesis. Although unlikely, it is possible that in the absence of ER, receptor
negative breast tumors will still develop in AIB1 transgenic mice.
AIB1 transgenic mice have been crossed with our ER fl/fl mice and AIB1/ER fl/+ mice were obtained. Currently, we are in the process of generating AIB1 homozygous & ER
fl/fl mice.
B. The anti-inflammatory effect of ERα in AIB1-induced mammary
tumorigenesis
Chronic inflammation is a major risk for various cancers (Karin, 2006). Specific
examples of inflammatory factors are TNFα and IL-6 which are known to promote tumor
growth. However, estrogen is known to inhibit inflammation by suppressing NF-κB
154 signaling and expression of pro-inflammatory cytokines and growth factors in
macrophage and tumor cells (Harnish, 2006; Steffan et al., 2006). ERα was shown to
mediate the anti-inflammatory effects of E2 in macrophages and mammary epithelial
cells (Harkonen and Vaananen, 2006). The increased occurrence of ER positive breast
cancer after menopause coincides with the drop of circulating estrogen level, indicating
that release of the inhibition on inflammation may affect breast cancer development. In addition, low ER expression is seen with inflammatory breast cancer, characterized by an aggressive phenotype and inflammatory symptoms. Presumably, since the ER positive breast cancer cells cannot efficiently produce TNFα and IL-6 to attract monocytes and macrophages in the presence of E2; macrophages and monocytes can not generate the cytokines to amplify inflammatory signals, due to the similar inhibition by E2. Most ER positive breast cancers are low-grade cancers and have slow progression (Holst et al.,
2007; Montano et al., 1999; Sorlie et al., 2003). In accordance with human breast cancers, most tumors in AIB1 transgenic mice are ER positive and have a latency of 9-16 months
(Torres-Arzayus et al., 2004). Therefore, we hypothesize: The long latency of the breast
cancer in AIB1 transgenic mice is due to the inhibitory effect of estrogen on
inflammation through ERα.
To test our hypothesis, AIB1 transgenic mice will be treated with pro-inflammation drugs,
TNFα and IL-6, or transplanted with ER null bone marrow to accelerate the tumor
progression. ER will be specifically knocked out in both mammary epithelial cells and
inflammatory cells in the AIB1 transgenic background. ER will also be deleted with
Adeno-Cre (or dox administration) in ER positive cancer cells derived from AIB1
transgenic mice.
155 We predict that TNFα and IL-6 treatment in AIB1 transgenic (tg) mice and ER null bone
marrow transplantation will accelerate the development of breast cancer. The pro-
inflammation drugs should produce similar effects on AIB1 promoted tumors. Specific
knockout of ER in monocytes and macrophages (tumor-associated macrophages (TAMs))
will inhibit the growth of breast cancer. The pro-inflammation treatment may accelerate
tumor progression by facilitating the ER positive breast tumor to break the barrier of
tumor suppressive basement membrane and myoepithelial cells resulting in invasiveness.
C. The role of ERα in mammary stem cells and epithelial progenitor cells
The mammary ductal network can be repopulated with a single mammary stem cell
(Shackleton et al., 2006; Stingl et al., 2006). Some adult mammary stem cells might be
ER-positive (Clarke et al., 2005). In accordance with this observation, ER null mice
exhibited rudimentary ductal networks but had no postnatal mammary gland development
(Mallepell et al., 2006). Therefore we hypothesize that ER is required for self-renewal
and proliferation of adult mammary stem cells.
To test our hypothesis, we will first determine whether the mammary stem cells defined
by Shackleton et al. (2006) and Stingl et al. (2006) are ER-positive. Then, we will
determine if ER is required for the stem cells to re-populate the mammary arbor by
deleting ER from the GFP labeled mammary stem cells and transplant the ER null stem
cells into mammary fat pad. Finally, we will determine whether the autocrine or
paracrine ER signal is required for stem cells to re-populate the mammary ductal network.
To determine whether paracrine ER signaling is required for mammary gland development, some differentiated ER positive epithelial cells will be mixed with GFP
156 labeled ER null mammary stem cells for the transplantation and vice versa. We predicted that ER autocrine signal is required for the mammary stem cells to re-populate the mammary arbor.
157
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