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This is to certify that the
thesis entitled

Identification of an Estrogen Receptor
Variant in Estrogen independent and/or
Tamoxifen Resistant Human Breast Cancer
Cell Lines

presented by

Feng Han

has been accepted towards fulfillment
of the requirements for

Master Wgee in Microbiology

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Date 5/1112101

0-7639 MS U is an Affirmative Action/Equal Opportunity Institution

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6/01 cJClRCIDanuo.p65-p.15

IDENTIFICATION OF AN ESTROGEN RECEPTOR VARIANT IN ESTROGEN
INDEPENDENT AND/OR TAMOXIFEN RESISTANT HUMAN BREAST
CANCER CELL LINES
By

Feng Han

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Department of Microbiology and Molecular Genetics

2001

ABSTRACT

IDENTIFICATION OF AN ESTROGEN RECEPTOR VARIANT IN ESTROGEN
INDEPENDENT AND/OR TAMOXIFEN RESISTANT HUMAN BREAST CANCER
CELL LINES

By

Feng Han

Estrogen receptor is important in the development of breast cancer, but a role for
it in the development of antiestrogen resistance has not been established. In this study, an
ER variant, ERAE3, was observed in two naturally selected cell lines that are estrogen
independent and/or tamoxifen resistant. The presence of ERAE3 decreased overall ER
function, as indicated by a 3-5 fold reduction of ERE activity in these cells. ERAE3 was
ectopically expressed in MCF-7 and LCCl cells, and the phenotype of transfectants was
examined by anchorage independent proliferation assays. It was observed that expression
of ERAE3 inhibited proliferation and was not sustainable in MCF-7 cells. In contrast, it
could be maintained for at least 15 passages in LCCl cells, where it did not detectably
inhibit proliferation. Expression of ERAE3 did not alter tamoxifen sensitivity in either
cell line. These results suggest that ERAE3 competitively inhibits wild-type ER activity
and negatively regulates cell proliferation. They further suggest that a change has
occurred in LCCl cells that both permits the development of estrogen independence and
allows the sustained expression of ERAE3, and that additional changes may lead to

tamoxifen resistance.

To Joy

iii

ACKNOWLEDGMENTS

I would like to thank Dr. Susan E. Conrad for her continuous support,
understanding, and inspiring guidance. I would also like to thank Dr. Richard Miksicek

for his great help. Without them, this work would not have been possible.

Specially thanks to the professors on my committee, Dr. Walter Esselman, Dr.

Karen Friderici, Dr. Richard Schwartz, for their insightful suggestions.

Many thanks to my current and previous labmates, Shibani Mukherjee, Andrew
Skildum, Yuping Tang, Hemant Vanna, and Cunjie Zhang. I appreciate your tremendous

help very much and have really enjoyed the time we work together.

TABLE OF CONTENTS

LIST OF FIGURES ................................................................................ vii
CHAPTER 1

Literature Review ..................................................................................... 1

1. Introduction ....................................................................................... 1

1.1. Breast cancer and estrogens ............................................................... 1

1.2. Endocrine therapy and antiestrogen resistance ......................................... 1

1.3. Tamoxifen resistance ...................................................................... 2

2. Molecular biology of estrogen receptor ....................................................... 3

2.1. The role of estrogen receptor in breast cancer .......................................... 3

2.2. Isofonns of estrogen receptor ............................................................ 3

2.3. Structure of estrogen receptor ............................................................ 4

2.4. Molecular mechanism of transcription activation by estrogen ...................... 4

2.5. Coregulators of estrogen receptor ........................................................ 6

3. Estrogens, antiestrogens, and selective estrogen receptor modulators .................... 7

3.1. Categorization of compounds bound to estrogen receptor ........................... 7

3.2. Chemical structures of estrogens, antiestrogens, and selective estrogen receptor
modulators ................................................................................... 8
3.3. Molecular basis of antiestrogen action ................................................... 8

3.4. Molecular basis of tissue Specificity of selective estrogen receptor modulators. . .9

4. Estrogen Receptor Variants ................................................................... 10
4.1. The presence of estrogen receptor variants in breast cancer ........................ 10
4.2. Paradoxical roles of estrogen receptor variants in the development of breast

cancer and antiestrogen resistance ...................................................... 10

5. Estrogen receptor downstream signaling .................................................... 12
5.1. Oncogenes and tumor suppressor genes regulated by estrogen ..................... 12
5.2. Growth factors and growth factor receptors regulated by estrogen ................. 13

6. Possible molecular mechanisms of tamoxifen resistance ................................. 14
6.1. Estrogen receptor and its variants ...................................................... 14
6.2. Estrogen receptor coregulators .......................................................... 16
6.3. Growth factors ............................................................................. l7

7. In vitro models of tamoxifen resistance—The LCC series ................................ 19

References ............................................................................................ 21

CHAPTER 2

Impaired ER Function in Estrogen Independent and/or Tamoxifen Resistant Human

Breast Cancer Cell Lines ........................................................................... 32
Introduction ........................................................................................... 32
Materials and Methods .............................................................................. 36
Results ................................................................................................. 37
Discussion ............................................................................................ 54
References ............................................................................................ 57

vi

LIST OF FIGURES

Figure 1.1 Structure of the estrogen receptor and the estrogen receptor

variant ERAE3 ......................................................................... 5
Figure 1.2 Chemical structures of estrogens and antiestrogens .............................. 9
Figure 2.1 Decreased ER function in LCCl and LCC2 cells ................................ 43
Figure 2.2 Expression of ER in MCF-7 derivatives and identification of

a 61 KD ER variant in LCC2 cells ................................................. 46
Figure 2.3 Identification of ERAE3 expression in LCC2 cells ............................... 47

Figure 2.4A. Verification of ERAE3 expression in pCDNA3-ERA3 transfected
MCF-7 and LCCl cells .............................................................. 48

Figure 2.4B. Loss of ERAE3 expression in stably transfected MCF-7 cells and
sustained expression of ERAE3 in stably transfected LCCl cells .............. 48

Figure 2.5A. Verification of ER and ERAE3 expression in stably transfected
LCCI cells ............................................................................. 52

Figure 2.5B.C. Effect of ER and ERAE3 expressions on the anchorage
independent proliferation of LCCl cells .......................................... 52

vii

CHAPTER 1

Literature Review

1. Introduction

1.1. Breast cancer and estrogens

Breast cancer is the most common type of cancer among women with an annual
worldwide incidence of one million. In western countries, the rate is especially high. One
out of every eight women will develop breast cancer in her lifetime.

Estrogen plays several important roles in the development of the reproductive tract
and secondary sex organs. These roles are reflected in the development of mammary
gland, regulation of the estrus cycle, and control of lactation. Estrogen also exerts effects
on the bone, liver, and cardiovascular systems.

Estrogen also plays important roles in the development and progression of breast
cancer. In the late 1890’s, it was observed that ovariectomy in cases of premenopausal
breast cancer led to tumor regression. In athymic nude mice xenografied with human
breast cancer cells, tumor formation and growth require the presence of estrogen( 1 ).
Through experimental models of carcinogen-induced mammary carcinoma, studies have
shown that estrogen is essential for the initiation and progression of breast tumors(2).
Based on this pivotal role of estrogen in the initiation and development of breast cancer,

endocrine therapy has been used in the treatment of breast cancer.

1. 2. Endocrine therapy, estrogen independence, and antiestrogen resistance

Current primary therapies for breast cancer include excision surgery, cytotoxic
chemotherapy, and radiation therapy. After these treatments, endocrine therapy has been
developed as the major adjuvant treatment for breast cancers that are responsive to
estrogen. The general principle of endocrine therapy is to inhibit the mitogenic stimulus
generated from estrogens. Antiestrogens have been developed for this purpose. Having
similar structures to estrogen, antiestrogens act through their competitive antagonism of
estrogen. Currently, several types of antiestrogens have been developed, including
nonsteroidal and steroidal. Among these, tamoxifen (a non-steroidal antiestrogen) has
been the most widely used and clinically proven effective(3).

Following endocrine therapy, most breast tumors undergo remission. However, many
breast tumors that initially respond to endocrine therapy eventually become estrogen
independent, and the patient relapses with acquired resistance to antiestrogens. In fact,
the development of multiple drug resistance in advanced breast cancer is the primary

reason for failures of current breast cancer therapies(3).

1.3. Tamoxifen resistance

Tamoxifen treatment is the most commonly used endocrine therapy in horrnone-
responsive breast cancer, especially as adjuvant therapy after removal of the primary
tumor(4). In fact, the application of tamoxifen clinically has been very effective in
decreasing both disease progression and the mortality rate. Worldwide clinical trials
indicate that the 5- and 10-year mortality rates of breast cancer patients can be reduced

20-25% by tamoxifen treatment(5). Nevertheless, the selective grth pressure by

tamoxifen often gives rise to tamoxifen resistance in many patients under tamoxifen

treatment. Tamoxifen resistance is the most significant problem of endocrine therapy.

2. Molecular biology of estrogen receptor

2.1. The role of estrogen receptor in breast cancer

Estrogen functions by binding to the estrogen receptor (ER). ER belongs to the
nuclear receptor superfamily of ligand-inducible transcription factors. ER has important
effects on the differentiation and maintenance of diverse tissues, including neural,
skeletal, cardiovascular, and reproductive(6, 7).

Based on the close correlation between estrogen and breast cancer, the importance of
estrogen receptor in the development of breast cancer has been established. Studies have
shown that nearly 70% of primary breast cancers have measurable ER expression while
only 7% of the epithelial cells in the normal breast tissue have the same level of ER

expression(8).

2. 2. Isoforms of estrogen receptor

There are two isoforms of ER, ER, and ERB. The two isoforms of ER share many
similarities. Both ERG and ERB respond to estrogen and stimulate transcription from
promoters containing estrogen responsive element (ERE) (9). Their functions are also
regulated by coregulators (10). While both ER0L and ERB have the same binding affinity
for estrogen, they are regulated differently by estrogen and may play different roles in

gene regulation. For example, depending upon cell types and promoter context, estrogen

can activate transcription from an AP-l Site with ERG; while it inhibits AP-l activity
with ERB. In addition, compounds that act as antiestrogens with ER“, including
tamoxifen, raloxifene, and ICI are potential activators with ERB at an AP-l site(11). The
current understanding of ERB fimction is mostly limited to brain and ovary tissues(12-l4).
In breast tissues, most work has been done towards clarifying the mechanisms by which
ER“ mediates the proliferation effects of estrogen. In the following discussions, ER

refers to ERG unless specified otherwise.

2. 3. Structure of estrogen receptor

ER has a structure typical of nuclear receptors. It is composed of six structural
domains, A-F (Figure 1). Its functional domains include two transactivation domains
(AF -1 and AF -2), a DNA binding domain, a ligand binding domain (LBD), and a hinge
region. AF -1 is located at the N-terminus and AF-2 is within the LBD. The LBD domain
is located in the C-terminal region of ERa. The structure of ER is important for its
binding to estrogens and antiestrogens, and its subsequent interactions with co-regulators,

which determines its effects on gene transcription.

MRNA 1 2 3 4 5 6 7 8

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1 1 85 250 31 1 551 595
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Figure 1.1 Structure of the estrogen receptor and the estrogen receptor variant

ERAE3.

2. 4. Molecular mechanism of transcription activation by estrogen

Currently, there are two known modes of ER function. Without the presence of
estrogen, ER exists in the form of complex with several heat shock proteins in the
nucleus. When bound by estrogen, ER undergoes an activating conformational change
and dissociates from the complex(15). In the “classical mode”, estrogen binds to the
ligand-binding domain and induces ER dimerization. Dimerized ER binds to estrogen
response elements (ERES) in the 5’ promoter region of target genes and activates
transcription of these genes(16). The second mode does not require ER dirnerization or
DNA binding by ER. After estrogen binds to ER, the complex can activate transcription
by protein-protein interaction with other transcription factors like c-Fos and c-Jun at AP-

] sites(l 1).

ER mediates transcription activation through its two transactivation domains, AF -1
and AF -2. AF -1 is activated by growth factors through the MAP kinase pathway(17);
while AF -2 responds to ligand binding (18). Transcriptional activation by both AF -1 and
AF-2 domains relies on specific promoter sequences, and may be tissue specific. AF -1
and AF -2 can function independently from each other; but optimum activity requires the
activation of both domains(l9).

Estrogen binds to ER through LBD (ligand binding domain). The crystal structure of
the human ER LBD, and how ligand binding affects this structure have been
established(20). After ligand binds to the LBD, it induces complex conformational
changes in and around the ligand-binding pocket, resulting in a repositioning of Helix 12,
the most C-tenninal helix of ER. The repositioning of Helix 12 plays an essential role in
the activation of AF -2 and the subsequent recruitment of co-regulators(21-24). When ER
LBD is bound by agonists such as estrogen, the conformation of the repositioned Helix
12 allows coactivators such as GRIPl to bind. When ER LBD is bound by antagonists,
different conformational changes occur, and coactivators can not bind to Helix 12. In
turn, transcription cannot be activated(25, 26). Therefore, the conformation of ER resulted

from ligand binding is essential for its function in transcriptional activation.

2. 5. Coregulators of estrogen receptor
ER transcriptional activity is regulated by various coactivators and corepressors. The
result of the interactions between ER and its coregulators is dependent upon the Specific

ligand bound to ER. Different ligands result in different conformations of ER, resulting in

the recruitment of different co-regulators, which could in turn regulate different subsets
of estrogen-responsive genes.

Coactivators play an important role in the ligand-dependent activation of transcription
by estrogen receptor. Agonists promote coactivator binding, while antagonists inhibit
coactivator binding(20). Transcriptional coactivators are a group of proteins that can
enhance ligand-dependent transcriptional activation by nuclear receptors(27, 28). After
binding to ligand-bound nuclear receptors, coactivators facilitate the recruitment of the
pre-initiation transcription complex and the activation of gene transcription. Coactivators
recognize agonist-bound nuclear receptor LBDs through the NR box, a short sequence
motif, LXXLL (L represents leucine and X represents any amino acid). These
coactivators include GRIPl/TIF-2/N-CoA2(29-31), SRC-I/N-COA1(25, 32, 33),
p/CIP/RAC3/ACTR/AIB1(31, 34, 35), and CBP/p300(36). Mutations at the NR box could
lead to the loss of affinity of coactivators for nuclear receptor LBDs(37). After
coactivators bind to estrogen-ER complex, transcriptional activation of estrogen-
responsive genes can be further stimulated. It has been Show that SRC-l ’8 binding to
estrogen-ER complex could increase histone acetyltransferase activity(38), which
functions to relax promoter DNA structure and facilitate the initiation of transcription.

ER can also recruit many corepressors including SMRT and N-CoR and negatively
regulate transcription(39, 40). ER-ligand complexes containing corepressors such as N-
CoR can increase histone deacetylase activity(3 8), which would be expected to inhibit
transcription (41).

The effects of coregulators on ER-mediated transcription can be very complex. For

example, SRC-l enhances the agonist activity of 4-OH TAM, and also interacts with

other coactivators synergistically to stimulate ER-dependent gene transcription(40, 42).
While N-CoR inhibits the agonist activity of 4-OH TAM, it cannot bind to the ICI
182,780-ER complex(43). The ultimate effects of coregulators on ER-mediated
transcriptional activation may depend on specific ligands bound by ER, various promoter

contexts, and different cell types.

3. Estrogens, antiestrogens, and selective estrogen receptor modulators

3.1. Categorization of compounds bound to estrogen receptor

The ER LBD can recognize compounds of different sizes, shapes, and chemical
properties. This ability enables a variety of compounds to be developed in order to
modulate ER transcriptional activity. These compounds, termed estrogen receptor
modulators, have been used to treat breast cancer, osteoporosis, and cardiovascular
disease. Based on their different behavior in transcriptional activation, they can be
divided into three categories- pure agonists, such as the endogenous estrogen 17‘3-
estradiol and the synthetic nonsteroidal estrogen diethylstilbestrol (DES); pure
antagonists, such as ICI 164,384; and selective estrogen receptor modulators (SERMS)
including tamoxifen and raloxifene(RAL).

While pure agonists stimulate and pure antagonists inhibit ER activity, SERMS can
act as either agonists or antagonists depending on specific tissue and promoter
contexts(44). For example, tamoxifen has tissue-Specific effects. It is known to
antagonize the effects of estrogen on mammary tissue, but acts as an estrogen agonist in
bone and the cardiovascular system. While tamoxifen reduces breast cancer incidence by

45% in high-risk women(45), it also increases the incidence of endometrial cancer (45,

46). The increased risk of endometrial cancer for patients treated with tamoxifen is a
result of tamoxifen’s effect as an agonist in the uterus. Another SERM, raloxifene,
functions entirely as an antagonist in the same tissue(46).

The mechanism of tissue Specific actions of SERMS is not clear. One possible
explanation is that specific conformations of the ER-ligand complex may be determined
by the molecular structures of different ER modulators. The resulting difference in the
conformation of the ER-ligand complex might determine different transactivation

properties, which eventually results in tissue selectivity (15, 16, 44).

3. 2. Chemical structures of estrogens, antiestrogens, and selective estrogen receptor
modulators
The chemical structures of commonly seen estrogens, antiestrogens, and SERMS are

listed in Fig. 2.

I10 “' (CH2)1oCON(nBu)Me
Tam Rel '01

Figure 1.2 Chemical structures of estrogens and antiestrogens.

3. 3. Molecular basis of antiestrogen action

Antiestrogens inhibit cell proliferation primarily by acting as antagonists to estrogen.
For example, Tamoxifen is a nonsteroidal triphenyl ethylene and has a similar structure
as estrogen. It acts as a competitive inhibitor of estrogen by binding to ER. When bound
by tamoxifen, ER can still dimerize, but cannot bind to ERES and hence gene
transcription cannot be activated. However, different antiestrogens might exert their
antagonism differently. For example, ICI has a higher ER binding affinity than
tamoxifen. When bound by ICI, ER cannot dimerize and therefore DNA binding and
transcriptional activation cannot be initiated. Furthermore, in addition to its inhibitory
effect through competitive binding to ER, ICI also significantly reduces the stability of
ER, leading to a rapid decrease in ER protein level and transcriptional activity(47). It is
also possible that different antiestrogens regulate different subsets of genes. It was
demonstrated that different antiestrogens, including 4-OH TAM, idoxifene, raloxifene,
GW7604, and ICI 182,780 induce distinct ERor-ligand conformations. These distinct
conformations could be recognized differentially by the transcription machinery,

resulting in different down-stream signaling(48).

4. Estrogen Receptor Variants

4.1. The presence of estrogen receptor variants in breast cancer

The ER gene has eight coding exons which generate the 6.2 kb wild-type ER mRNA.

Abberant mRNA splicing can produce different forms of ER mRNA with single or

multiple exon deletions from exon 2 to exon 7. The presence of these ER variant

10

mRNAs have been widely detected in many breast cancers varying widely in terms of
their proportion to wild type ER mRNA and in different studies(49-57). The significance
of these ER variants has been studied both for diagnostic purposes and for understanding
antiestrogen resistance. Among these ER variants, the most common and extensively
studied ones include exon 3 deletion (ERAE3) and exon 5 deletion (ERAES).

Exon 3 codes for the second zinc finger of the DNA binding domain. The ERAE3
therefore does not have a functional DNA binding domain. Consequently, the ERAE3
protein cannot bind to ERES and therefore cannot activate ERE activity in transient
transfection assays. Studies also confirmed this. When mixed with wild type ER at a 1:1
ratio, ERE activity was inhibited by 30%, suggesting that ERAE3 functions as a dominant
negative ER variant (51). Furthermore, ectopic expression of ERAE3 in estrogen
responsive human breast cancer cells suppressed their in vivo invasiveness(58). Exon 5
encodes for part of the ligand binding domain. The ERAES lacks both a functional LBD
and the second transactivation domain AF-2. Unlike ERAE3, ERAES cannot dimmerize
with both wild type ER and ER variants. It has been observed that increased levels of

ERAS mRNAs are present in some breast tumor tissues(49).

4. 2. Paradoxical roles of estrogen receptor variants in the development of breast
cancer and antiestrogen resistance
The role of ER variants in the development of breast cancer and antiestrogen
resistance has been widely studied and controversial. Using sensitive techniques such as
RT-PCR, many variant ER mRNAs have been detected in normal breast tissues, breast

cancer cell lines, and clinical breast tumor samples. Some of these ER variants were

11

shown to arise from abberant mRN A splicing. However, the origin and consequence of
these variants are not clear. Most breast tumors express wild-type ER at the same time as
they express mutant ER. Some researchers showed the presence of ER variant mRNAs
in more than 30% of breast tumors and the rate increased with the emergence of
tamoxifen resistance, while others failed to observe any evidence of ER variants in in
vitro derived tamoxifen resistant cell lines(59-61). Furthermore, overexpression of ERAE3
or ERAES is not sufficient to induce tamoxifen resistant phenotype in MCF-7 cells(57).
Although there were small proportion of ER variants with protein produced, whether the
majority of the ER variants are expressed and translated into a large amount of proteins
that result in different activities on antiestrogen resistance is not known (62).

The effects of different ER variants on ER-regulated gene transcription are likely to
be complex. Some variant ERS such as ERAE3 have been Shown to be dominant negative

ER mutants that substantially inhibit transcription at ERES, while others such as ERAES

can constitutively activate transcription at an ERE site in yeast (49). Even for ERAE3,
there is evidence that it can activate transcription at AP-l site in transient transfection
assays(63). While ER variants with exon deletions have been widely detected, other types
of mutant ERS have also been observed. An ER mutant can use tamoxifen as an agonist
in some MCF-7 cell derivatives (64). While most ER variants show decreased responses
to estrogen, a mutation affecting the border of the hinge and LBD enabled increased
sensitivity to estrogen compared to wild-type ERor. The ectopic expression of this ER
variant increased cell proliferation in stably transfected breast cancer cells and enhanced
TIF-2 binding at low levels of estrogen. In addition, a significant proportion (34%) of

hyperplasias, a type of early premalignant lesion, had such mutations(65). Because most

12

cancers arise from premalignant lesions, the existence of such ER variants in those
premalignant lesions indicates a possible importance of ER variants in the development
of breast cancer. While some ER variants exhibit some fimctional properties in breast
cancer cell lines, it is not known that whether these effects would occur in a Significant

proportion of breast cancer patients.
5. Estrogen receptor downstream signaling

5.1. Oncogenes and tumor suppressor genes regulated by estrogen

Because of the importance of oncogenes in carcinogenesis, the relationship between
estrogen and oncogenes has been extensively studied. Oncogenes that are regulated by
estrogens and antiestrogens include c-myc, c-fos, and c-jun. It was found that the
transcription of c-myc and c-fos was directly regulated by estrogen in breast cancer cell
lines(66). When antisense c-myc oligonucleotides were introduced into MCF-7 cells, the
proliferation of the cells in the presence of estrogen was inhibited(67). Through ER, c- I
myc expression is regulated by estrogen and is also required for estrogen-induced

proliferation.

5. 2. Growth factors and growth factor receptors regulated by estrogen

Many growth factors and growth factor receptors have been identified to be involved
in breast cancer initiation and development (68, 69). For example, Overexpression of the
c-erb-B2 gene is detected in approximately 25% of human breast tumors (70). Increased

IGF-II mRNA or protein has also been observed in breast cancers (71). In addition,

13

overexpression of EGF can induce MCF-7 cells to form tumors in ovariectomized nude
mice. Although overexpression of growth factors is not essential to breast cancer
development, antisense to EGF-R reduces the tumorigenicity of three breast tumor
models (72).

It has been demonstrated that the expression of many growth factors and their
receptors are regulated by estrogen. These estrogen-responsive growth factors include
Epidermal Growth Factor (EGF), Transforming Growth Factors (TGFoc, TGFB), c-erb-
B2, and Insulin-Like Growth Factors (IGF-I and IGF-II). The subsequent effects of these
growth factors on cell proliferation are associated with growth factor receptors such as
EGF receptors (EGFR) and IGF receptor (IGF-IR).

Presumably through ER, studies have Shown that estrogen increases expression of
growth factors. Increased levels of growth factors and/or decreased levels of grth
factor inhibitors were found in estrogen treated breast cancer cells (73-77). Estrogen
increases EGF-R expression in hormone responsive tissues(78). In some cells, IGF-II
expression can be induced by estrogen(79). The stimulatory effect of EGF on
proliferation can be inhibited by tamoxifen (80, 81), suggesting the up-regulation of
growth factors by estrogen is mediated through ER and could be reversible. In addition,
cross talk exists between growth factors and estrogen-regulated genes. It has been
Shown that p82, an estrogen-responsive gene, is up-regulated by TGF.

In conclusion, some of growth factors are up-regulated by estrogen. The interactions
between those growth factors and other estrogen regulated genes are likely to be

complex.

14

6. Possible cellular and molecular mechanisms of tamoxifen resistance

Understanding the mechanism of tamoxifen resistance is critical to the development
of new drugs and to improving the treatment of breast cancer patients. Several possible
mechanisms have been proposed to account for antiestrogen resistance including:
changes in pharrnacokinetics, abnormal ER functions, overexpression of growth factors
or their receptors (82-84). This review will address recent studies on ER signaling and

overexpression of growth factors.

6.]. Estrogen receptor and its variants

Many breast tumors that initially respond to tamoxifen eventually recur with acquired
resistance. However, most tamoxifen resistant patients are still sensitive to ICI treatment,
an alternative form of endocrine therapy. This indicates that the ER is still involved in
promoting tumor growth(83). By immunohistochemical assay, it was found that ER
expression was reduced, but still maintained, in tamoxifen resistant tumors(85).

ER can interact with the activator protein-l (AP-1) transcription factor complex
through protein-protein interactions that are independent of ER DNA binding and, in
certain ER-positive cells, this may allow TAM to exert an agonist activity on AP-l-
regulated genes. One AP-l activating enzyme is c-Jun NH2-terminal kinase (JNK). In
human breast tumors with acquired tamoxifen resistance, increased AP-l DNA binding
and JN K activity was observed(86).

Changes in ER function and subsequent signaling could also be mediated by ER

mutants or variants, since such variants could affect the ability of tamoxifen to form

15

complexes and/or regulate the activity of ER. Various ER variants have been observed in
cell lines selected in the presence of antiestrogens in vitro, as well as in tamoxifen
resistant tumor samples(87). While some studies found that ER variants such as ERAE3
and ERAES were present at higher levels in tamoxifen resistant breast tumors, the ectopic
expression of those variants alone failed to confer estrogen independence and/or
tamoxifen resistance to estrogen-responsive breast cancer cells(57, 58).

It has also been speculated that the interaction between EROt and ERB plays a role in
tamoxifen resistance. ERor and ERB can form heterodimers, and 4-OH TAM can activate
transcription through these heterodimers in the context of specific promoters and cell
types(88, 89). This suggests that with both ERB and ERor present in breast cancer cells, 4-
OH TAM could act as an agonist instead of an antagonist in terms of cell proliferation.

Interestingly, some estrogen independent and/or antiestrogen resistant cell lines still
express wild-type ER at significant levels(64). In such cases, it was suggested that other
factors, including ER coregulators and growth factors, might be involved in causing

estrogen independence and antiestrogen resistance.

6. 2. Estrogen receptor coregulators

The importance of coregulators in ER-mediated transcription suggests a hypothesis
that changes in the expression or recruitment of coregulators could lead cells to
tamoxifen resistance (40, 90). While the estrogenic activity of 4-OH TAM can be
increased by coactivators such as L7/ SPA (43), it can be inhibited by corepressors such as
SMRT (40). In tamoxifen resistant MCF-7 xenografts in nude mice, the expression of N-

CoR, an ER corepressor, was reduced(91). These findings suggest that both the

16

elimination of corepressors and recruitment of coactivators could confer tamoxifen
resistance to cells. However, there is also evidence that is contradictory to this
hypothesis. Studies have shown that overexpression of SRC-l in MCF-7 cells does not
induce changes in their response to 4-OH TAM(92). A close examination of several
tamoxifen resistant cell lines and a number of tamoxifen resistant human breast tumors
revealed no change in the expressions of TIP -1, RIP 140, or SMRT (93). The effect of ER

coregulators on tamoxifen resistance therefore still needs to be further studied.

6. 3. Growth factors

Changes in the signaling pathways that regulate cell proliferation and apoptosis could
also influence ER-regulated gene networks and confer tamoxifen resistance on cells. AS
discussed previously, growth factors can affect ER function and participate in the ER-
regulated gene network. The changes in growth factor expression and Signaling can
reduce the requirement for estrogen stimulation of cells to proliferate, giving cells
phenotypes such as estrogen independence and/or tamoxifen resistance. In such cases,
estrogenic signaling pathways from the ER could still remain intact in those cells.

There is abundant evidence that growth factors are involved in the development of
estrogen independence and tamoxifen resistance. The initial evidence of growth factor
involvement in tamoxifen resistance came in the 1980’s. In several horrnone-
independent breast cancer cell lines, a number of growth factors were expressed at high
levels and were no longer regulated by hormones(69, 76). Subsequently, a variety of
growth factors have been examined in tamoxifen resistant cell lines and tumors. TGF-or

was found to be constitutively expressed in many estrogen-independent cells (76, 94).

17

Estrogen-independent breast cancer cell lines also expressed high levels of EGF-R
relative to hormone-dependent cells (95, 96). IGF-I, TGF-or, and PDGF were found to be
secreted constitutively by honnone- independent human breast cancer cells and their
activities can induce the formation of estrogen-independent tumors in MCF-7 cell-
xeografted ovariectomized athymic nude mice (80). Some researchers have reported an
increase in the levels of IGF-II in tamoxifen treated patients(97, 98).

Studies on the effects of growth factors on tamoxifen resistance have either
overexpressed growth factors or inhibited growth factor functions. While overexpression
of some grth factors or growth factor receptors confers estrogen independence and/or
tamoxifen resistance, others fail to change the phenotype of the cells. Overexpression of
TGF-B has been found in human breast tumors resistant to tamoxifen. By introducing
TGF-B antibodies into tamoxifen resistant breast cancer cells, it was found that the cells
lost their resistance to tamoxifen when inoculated into nude mice(99). Overexpression of
EGF and IGF-I partially reverse the growth inhibitory effects of antiestrogens (100).
Overexpression of IGF-I receptor moderately enhanced the growth responsiveness to
estrogen and reduced the estrogen growth requirement in MCF-7 cells(lOl). In addition,
overexpression of c-erb-B2 enabled estrogen responsive breast cancer cells to acquire
estrogen-independent growth and reduced responsiveness to tamoxifen (102-104). On the
other hand, the ectopic expression of TGF-or failed to induce hormone-independence in
MCF-7 cells(69). However, this does not exclude the possible roles of these growth
factors in the development of estrogen independence. Other recent studies provide more
evidence supporting this statement. It has been shown that by introducing antisense

TGF-or oligonucleotide into estrogen-responsive breast cancer cells, the estrogen

18

responses in those cells were reduced (105, 106). Overexpression of TGF-or was observed
in several tamoxifen resistant cell lines (99, 107), but introducing TGF antibodies into

those cells could not restore their tamoxifen responsiveness (99).

7. In vitro models of tamoxifen resistance—-The LCC series

The development of estrogen-independent cell lines has been widely used as
laboratory model to study the problem of clinically antiestrogen resistance clinically. For
estrogen-responsive cell lines, MCF-7, ZR-75-1 and T47D are commonly used. MDA-
MB-231 and MDA-MB-435 are ER negative cell lines. Studies using these cell lines have
provided much understanding of the mechanisms of how hormones regulate cell
proliferation.

My research utilized the MCF-7, LCC1, and LCC2 cell lines. MCF-7 cells were
originally isolated from a malignant pleural effusion in a postmenopausal breast cancer
patient, and have been widely used in breast cancer research. MCF-7 cells are estrogen
dependent and antiestrogen sensitive. Dr. Robert Clarke at the Vincent T. Lombardi
Cancer Center developed a series of MCF-7 derivatives which mimic the development of
estrogen-independent and antiestrogen resistant tumor grth observed in human breast
cancer patients. MCF-7 cells were inoculated into ovariectomized athymic nude mice and
selected in vivo for estrogen-independent growth. The circulating steroid hormone profile
of these mice is similar to that seen in postmenopausal women. LCC1 cells were isolated
and characterized as estrogen independent but antiestrogen sensitive. LCC1 cells mimic

some of the critical aspects of the early progression to a more aggressive tumor type--

Estrogen and progesterone receptor positive, steroid non-responsive, and increased
metastatic potential(108). LCC1 cells were further selected in vitro for growth in the
presence of triphenylethlene tamoxifen or ICI 182,780. LCC2 cells were isolated as
tamoxifen resistant but ICI sensitive(109), and LCC9 cells as both ICI and tamoxifen
resistant(110). The characteristics of LCC1, LCC2, and LCC9 cells were assessed,
including their in vitro proliferative capacity, in vivo tumorgenicity, and competitive
binding activities of steroid hormone receptors (109, 111). LCC1 cells proliferate well in
the absence of estrogen, but still sensitive to tamoxifen. LCC2 cells can proliferate in an
anchorage-independent manner and have significantly increased ability to form tumors in
nude mice in the presence of tamoxifen. While the proliferation of LCC2 cells is
inhibited by ICI, there is no inhibitory effect of ICI on the proliferation of LCC9 cells.
Both ER and progesterone receptor are present and are regulated by estrogen in MCF-7,
LCC1, LCC2, and LCC9 cells. LCC9 cells have a significantly elevated PR level
compared to other cell lines. The fact that the growth of LCC1 and LCC2 cells can still
be further stimulated by estrogen and estrogen-regulated genes such as p82 are still
regulated by estrogens and antiestrogens suggests the existence of functional ER in these
cells. The establishment of LCC1 and LCC2 cells is the first case of tumor formation
from MCF-7 cells grown in vivo without estrogen (112). Especially, the phenotype of
LCC2 cells (TAM resistant, but ICI senstitive) mimics a clinical pattern of tamoxifen
resistant patients that have undergone endocrine therapy. The pattern of metastasis
exhibited by these cells is very similar to that observed in breast cancer patients, and

hence these cells can be used as a model for the study of antiestrogen resistance.

20

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cellular effect against a choriocarcinoma cell line, Int J Oncol. 12: 1171-6., 1998.

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resistance of human breast carcinomas in vivo by neutralizing antibodies to
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inhibitory effects of antiestrogens on T 47D human breast cancer cell growth,
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growth requirements, enhance survival, and promote E-cadherin-mediated cell-
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Pietras, R. J ., Arboleda, J ., Reese, D. M., Wongvipat, N., Pegram, M. D., Ramos,
L., Gorman, C. M., Parker, M. G., Sliwkowski, M. X., and Slamon, D. J. HER-2
tyrosine kinase pathway targets estrogen receptor and promotes horrnone-
independent growth in human breast cancer cells, Oncogene. 10: 2435-46., 1995.

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cells overexpressing transfected c-erbB-2 have an in vitro growth advantage in
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sensitivity in vivo, Breast Cancer Res Treat. 34: 97-117., 1995.

Benz, C. C., Scott, G. K., Sarup, J. C., Johnson, R. M., Tripathy, D., Coronado,
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B., Normanno, N., Ciardiello, F., Day, A., Cutler, M. L., and et a1. Expression of
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Inhibition of estrogen-induced breast cancer cell proliferation by reduction in
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Brunner, N., Frandsen, T. L., Holst-Hansen, C., Bei, M., Thompson, E. W.,
Wakeling, A. E., Lippman, M. E., and Clarke, R. MCF7/LCC2: a 4-
hydroxytamoxifen resistant human breast cancer variant that retains sensitivity to
the steroidal antiestrogen ICI 182,780, Cancer Res. 53: 3229-32., 1993.

Brunner, N., Boysen, B., Jirus, S., Skaar, T. C., Holst-Hansen, C., Lippman, J.,
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antiestrogen-resistant MCF-7 variant in which acquired resistance to the steroidal
antiestrogen ICI 182,780 confers an early cross-resistance to the nonsteroidal
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Brunner, N., Boulay, V., Fojo, A., Freter, C. E., Lippman, M. E., and Clarke, R.
Acquisition of hormone-independent grth in MCF-7 cells is accompanied by
increased expression of estrogen-regulated genes but without detectable DNA
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human breast cancer cells, Clin Exp Metastasis. 11: 15-26., 1993.

31

CHAPTER 2

Impaired ER Function in Estrogen Independent and/or Tamoxifen Resistant Human
Breast Cancer Cell Lines

Introduction

Breast cancer is the most common type of cancer in women, with an annual
worldwide incidence of one million new cases. Studies have shown that estrogen is
essential for the initiation and progression of breast tumors (1, 2). Based on the pivotal
role of estrogen, antiestrogens have been developed as major drugs for the treatment of
breast cancer patients. Among them, tamoxifen is the most commonly used endocrine
therapy in hormone-responsive breast cancer, especially as adjuvant therapy after
removal of the primary tumor(3). The application of tamoxifen clinically has proved very
effective in both decreasing disease progression and reducing the mortality rate of breast
cancer. Worldwide clinical trials indicate that the 5- and lO-year mortality rates of breast
cancer patients can be reduced 20-25% by tamoxifen treatment (4). Nevertheless, many
breast tumors that initially respond to endocrine therapy become estrogen independent. In
addition, selective growth pressure gives rise to tamoxifen resistance in many patients
under tamoxifen treatment. In fact, the development of tamoxifen resistance in advanced
breast cancer is a primary reason for the failure of current breast cancer therapies.

Estrogen functions by binding to the estrogen receptor (ER), which has been shown to
play an important role in the development of breast cancer. Studies have shown that
nearly 70% of primary breast cancers have measurable ER expression, while only 7% of

the epithelial cells in normal breast tissue have detectable ER expression (5). There are

32

two isoforms of ER, ERor and ERB (6). In breast tissue, most work has been done
towards clarifying the mechanisms by which ERa mediates estrogen’s growth
stimulatory effects. In the absence of estrogen, ER exists in a complex with several heat
shock proteins in the nucleus. When bound to estrogen, ER undergoes an activating
conformational change, and dissociates from this complex (7). Currently, there are two
known modes of ER function. In the “classical mode”, estrogen binds to the ligand-
binding domain and induces ER dirnerization. Dimerized ER binds to estrogen response
elements (ERES) in the 5’ promoter region of target genes, and activates transcription of
these genes (8). The second mode does not require ER dimerization or DNA binding by
ER. After estrogen binds to ER, the complex can activate transcription by protein-protein
interaction with other transcription factors like c-Fos and c-Jun at AP-l sites (9).

The ER gene has eight coding exons, which generate the 6.2 kb wild-type ER mRNA.
Aberrant mRNA splicing can produce ER variant mRNAs with various exon
deletions(lO-18). The significance of these ER variants has been investigated for both
diagnostic purposes and for understanding antiestrogen resistance. The role of ER
variants in the development of breast cancer or antiestrogen resistance has been
controversial. By RT-PCR, variant ER mRNAs have been detected in normal breast
tissue, breast cancer cell lines, and clinical tumor samples. Although there were a small
proportion of samples where ER variant proteins were produced, whether the majority of
the ER variants are expressed and translated into protein is not known (15). Some
researchers showed the presence of ER splice variant mRNAs in more than 30% of breast
tumors and the prevalence of certain variants increased with the emergence of tamoxifen

resistance, while others failed to observe any evidence for ER variants in in vitro derived

33

tamoxifen resistant cell lines (18-20). Among the ER variants detected, one of the most
commonly seen is an exon 3 deletion (ERAE3). Exon 3 encodes the second zinc finger of
the DNA binding domain. The ERAE3 protein cannot bind to ERES and therefore cannot
activate ERE activity in transient transfection assays. When ERAE3 was mixed with wild
type ER at a 1:1 ratio, ERE activity was inhibited by 30%, suggesting that ERA3
functions as a dominant negative ER variant (17). Furthermore, ectopic expression of
ERAE3 in estrogen responsive human breast cancer cells suppressed their in vivo
invasiveness (21).

Understanding the mechanism of tamoxifen resistance is critical to the development
of new drugs and to improvements in the treatment of breast cancer patients. Tamoxifen
acts as a competitive inhibitor of estrogen by binding to ER. Several possible
mechanisms have been proposed to account for antiestrogen resistance including: loss of
ER, loss of ER cofactor(s), abnormal metabolism of tamoxifen, mutant ER, and
overexpression of growth factor receptors (22-24). Various ER mutations have been
observed in some of the cell lines selected in vitro for resistance to antiestrogens, as well
as in tumor samples (25, 26). However, many estrogen independent and/or antiestrogen
resistant cell lines still express wild-type ER at Significant levels (27), suggesting that
other mechanisms of resistance might exist.

The development of estrogen-independent cell lines has been used as a laboratory
model to study the problem of antiestrogen resistance. This study will utilize the MCF-7,
LCC1, and LCC2 cell lines. MCF-7 cells were originally isolated from a malignant
pleural effusion in a postmenopausal breast cancer patient, and have been widely used in

breast cancer research. MCF-7 cells are estrogen dependent and antiestrogen sensitive.

34

Dr. R Clarke et al at the Vincent T. Lombardi Cancer Center developed a series of MCF-
7 derivatives, which mimic the development of estrogen-independent and antiestrogen
resistant tumor growth observed in human breast cancer patients (28-31). MCF-7 cells
were inoculated into ovariectomized athymic nude mice and selected in vivo for estrogen-
independent growth. LCC1 cells were isolated and characterized as estrogen independent
but antiestrogen sensitive. Then, LCC1 cells were selected in vitro for growth in the
presence of tamoxifen, giving rise to LCC2 cells. LCC2 cells are tamoxifen resistant, but
still sensitive to the steroidal antiestrogens such as ICI 182,780. Among these MCF-7
derivatives, we are particularly interested in LCC2 cells because their phenotype is
Similar to those of breast cancer patients who develop tamoxifen resistance during
endocrine therapy. This study was aimed at identifying the biochemical changes
occurring during the development of estrogen independence and tamoxifen resistance in

these cell lines.

35

Materials and Methods

Cell culture

MCF-7 cells were obtained from Dr. Michael Johnson. LCC1 and LCC2 breast
cancer cells were obtained from Dr. Robert Clarke (Lombardi Cancer Center) and were
maintained in IMEM/FBS, i.e. IMEM media with 2mM glutamine (Biofluids, Inc.,
Rockville, MD) supplemented with 5% fetal bovine serum (Hyclone), penicillin (100
units/ml) and streptomycin (100 ug/ml) (Life Technologies). To study the effects of
estrogen and antiestrogens, cells were depleted of estrogen by plating in IMEM/CS8, i.e.
IMEM without phenol red and supplemented with 5% of charcoal-stripped fetal bovine

serum (Hyclone) (32).

Reagents and plasmids

17B—estradiol (E) and 4-hydroxytamoxifen (4-OH TAM) were purchased from Sigma.
ICI 182,780 (ICI) was purchased from AstraZeneca. ERAE3 and wild-type ER
expression vectors (pCDNA3-ERAE3 and pCDNA3-MERot) were constructed by
insertion of ERA3 and wild type ERor cDNAS into a G418 selectable plasmid vector
containing CMV promoter, pCDNA3 (Invitrogen). pBgal-Basic was purchased from
Clontech Laboratories. Lipofectin were purchased from Life Technologies. The ERE-
luciferase reporter construct, ERE2-tk109-luc, was obtained from Dr. Gehm at
Northwestern University Medical School (33).
Primers and PCR conditions

The primers used for RT-PCR were (5’-CTGCCAAGGAGACTCGCTAC-3’) as the

upstream primer and (5’-AAGGCACTGACCATCTGGTC-3’) as the downstream

36

primer. The primers used for genomic PCR were (5’-
CGCTCGAGTGGGGTGCAACGTAGTAAGA-3’) as the upstream primer and (5’-
GCGAATTCCAATGGGTAGAGCCAG-3’) as the downstream primer. PCR reactions
were performed under the following conditions. Five ug of total RNA and 10 pmol of
the downstream primer were denatured by incubation for 10 min at 65°C. Twenty u] of
reverse transcription mixture (50 mM Tris-HCl, pH 8.3, 40 mM KCl, 6 mM MgCl2, 1
mM DTT, 50 uM dNTPs, 200 units of MMLV-Reverse Transcriptase (Life
Technologies)) were incubated for 1 h at 37°C. Four pl of reverse transcription mixture
(or 1 pg of genomic DNA) was amplified in a final volume of 100 pl containing 250 nM
of each primer, 200 uM dNTPS, 1.5mM MgClz, and 2.5 units of TAQ DNA polymerase
(Life Technologies). Each RT-PCR consisted of 35 cycles (60 s at 62°C, 60 s at 74°C,
and 30 S at 94°C). Each genomic PCR consisted of 40 cycles (60 S at 58°C, 60 S at 74°C,
and 60 s at 94°C). PCR products were visualized on agarose gels stained with ethidium
bromide. The DNA fragments of interest were then recovered and purified using
GenElute Agarose Spin Column (Sigma), and sequenced at the Michigan State University

Sequencing Center.

Western blotting

Cell extracts were prepared as follows. Cell monolayers (80% confluent) were
washed twice in ice-cold PBS and collected by scraping and centrifugation. Cell pellets
were resuspended in ice-cold Lysis Buffer (50 mM HEPES, 150 mM NaCl, pH 7.5, 1

mM EDTA, 2.5 mM EGTA, 0.1% Tween-20, 10% glycerol, 1 mM DTT, 0.1 mM PMSF,

10 ug/ml leupeptin, 2 ug/ml aprotinin, 10 mM B-glycerophosphate, and 1 mM NaF) and

37

lysed by sonication on ice (Duty cycle 80%, output 6, 10 s) using a Sonifier 450 (Branson
Ultrasonics Corporation, Danbury, CT). The cellular debris was removed by
centrifugation (12,000g, 2 min, 4°C). The total protein concentration in the cell lysate
was measured using a Bradford Assay (Bio-Rad, Inc). Twenty pg of total protein fi'om
each sample was separated on a 12% polyacrylamide gel by SDS-PAGE and then
transferred to PVDF membrane (NEN Life Science Products) in transfer buffer (25 mM
Tris, 192 mM Glycine, and 15% methanol) using the XCELL gel transfer system (Novex
Experimental Technology). Western analysis of ER utilized a mouse monoclonal

antibody (Mab-17) that was raised against recombinant ERor protein and which

recognizes an amino-terminal epitope present in both wild-type ER and ERAE3. The
membrane was blocked in 5% dry milk in PBST (PBS + 0.1% Tween 20) and incubated
with a 1:2 dilution of the ER antibody in PBS at 4°C ovemight. The antibody was
removed and the membrane was washed with PBST three times for 10 min each. A
peroxidase-labeled secondary antibody (American Qualex, San Clemente, CA) was
diluted 1:2000 in 5% dry milk in PBST and incubated with the membrane for 1 h at room
temperature. Proteins were then visualized using Supersignal West Pico

Chemiluminescent Substrate (Pierce).

Transient transfection and Iuciferase assay

Cells were plated in IMEM/CSS at 4x105 cells per 60 mm tissue culture plate, and
incubated for two days to deplete them of estrogen. Five pg of ERE2-tklO9—luc and 1 pg
of pBgal-Basic were then co-transfected using Lipofectin (Life Technologies) as the

transfection agent. Cells were incubated with Lipofectin-DNA complexes in phenol red-

38

free IMEM media for 6 h. Media containing Lipofectin and DNA was removed, and
cells were washed twice in PBS and then incubated in IMEM supplemented with 5% CSS
with or without E (10'9M), 4-OH TAM (1045M), or ICI (10'9M). Treatments were added
as stock solutions in absolute ethanol, and ethanol was added to control media to the
same final concentration (0.1%) in all plates. Eight h after treatment, cells were washed
twice with PBS and harvested by scraping and centrifugation. Cell pellets were
resuspended in 200 pl of Reporter Lysis Buffer (Promega Corp., Madison, WI) and lysed
by freeze-thawing. The protein concentrations in the extracts were determined using the
Modified DC Assay (Bio-Rad). Aliquots of the lysate were used to assay B-galactosidase
activity according to manufacturer’s manual (34). Briefly, cell lysate was incubated with
Reaction Buffer Mixture (Clontech Laboratories) at room temperature for 1 h, and the B-
galactosidase activity was determined by measuring the light emitted from the mixture
using a Turner TD-20e luminometer. The Iuciferase assay was carried out with 20 pl of
lysate and 100 pl of Luciferase Assay Reagent (Promega). The light emitted was
measured for 10 S using a Turner TD-20e luminometer. The final luciferase activity was
expressed relative to the B-galactosidase activity in the same extract, and is shown as

means :I: SD of three independent transfection experiments done in triplicate.

Stable transfections

Stable cell lines were obtained by transfecting MCF-7 and LCC1 cells with
pCDNA3-ERA3 and pCDNA3-MER0L by Lipofectin. Cell lines were isolated in medium
containing 400 pg/ml G418. Single colonies were isolated by ring cloning and serial

dilution in 96-well dishes. Cells were subsequently maintained in medium containing 40

39

ug/ml G418. Clones expressing ERAE3 or wild-type ER were identified by Western

blotting of cell extracts.

Cell growth assay (soft agar assay)

Cells were plated in phenol red-free IMEM with 5% CSS for two days and then re-
plated at a density of 5X104 cells/well in soft agar in 6-we11 dishes. Briefly, cells were
suspended in IMEM with 5% CSS containing various treatments and 0.3% Agar Nobel
(Difco Laboratories, Detroit, MI), and plated on the top of IMEM with 5% CSS
containing treatments and 0.6% Agar Nobel. Cells were grown for 21 days, with feeding
every 5 days. Colonies were then stained with 0.1% neutral red and analyzed by
microscopy. Colonies larger than 60 pm (more than 50 cells) were counted, and six
independent wells per treatment per sample were averaged. The results were expressed as

mean +/- standard deviations.

RNA extraction and northern blot analysis

RNA was isolated using Trizol reagent and quantified by Spectrometry. Twenty pg
of total RNA was separated on 1.0% agarose/formaldehyde gels and transferred overnight
to nitrocellulose membrane (Schleicher & Schuell). Membranes were prehybridized in
prehybridization buffer (50% formamide, 5% dextran sulfate, 50 mM NaPO4, pH 6.8,
0.1% SDS, 3xSSC, and 5xDenhardt’S Solution) with 100 pg/ml sheared salmon sperm
DNA at 42°C for 2 h. Membranes were then hybridized to 107 dpm of 32P-labeled c-myc
cDNA probe. Blots were hybridized at 42°C for 16—24 h. Afier hybridization, blots were

subjected to two washes (0.2 X SSC, 0.1% SDS) at 42°C for 10 min each. Signal

40

intensities were measured using the Phosphorlmager system (Molecular Dynamics), and
blots were exposed to X-ray film at -80°C overnight. To normalize, blots were stripped

and probed with labeled human GAPDH.

4l

Results

ER activities in MCF 7, LCC1 and LCC2 cells

To identify the biochemical changes that are responsible for the estrogen
independence and/or tamoxifen resistance of LCC1 and LCC2 cells, their ER activities
were compared to that in MCF7 cells (Figure 2.1). MCF-7, LCC1, and LCC2 cells were
plated in medium containing 5% charcoal stripped serum (CSS) for two days to deplete
cells of estrogen. An ERE-luciferase plasmid construct was then transiently transfected
into cells along with a control B-galactosidase construct. After transfection, cells were
treated with E, 4-OH TAM, or ICI for 6 h, harvested and analyzed for luciferase activity
as described in Materials and Methods. The luciferase activities, normalized to B-
galactosidase activity, are Shown in Figure 1. In all three cell lines, ER activity was up-
regulated by E and down-regulated by 4-OH TAM and ICI, suggesting that the estrogen
independence of LCC1 cells and the tamoxifen resistance of LCC2 cells are not due to
changes in ER function. Interestingly, although ER was still responsive to E, 4-OH
TAM, and ICI, the maximum ER activity in LCC2 cells was 4-5 fold lower than that in
MCF-7 and 3-4 fold lower than that in LCC1 cells, suggesting some defect in ER levels

or function.

42

 

 

Adjusted Luciferase Activity

CSS E ICI TAM E+IC| E+TAM

Figure 2.1 Decreased ER function in LCC1 and LCC2 cells. ER function was
assessed by transient transfection of an ERE-luciferase construct into MCF-7 and LCC2
cells along with pBgal-Basic as a control. Cells were treated with E (10'9M), 4-OH TAM
(1045M), or ICI (10'9M). Six h later, cells were harvested and luciferase and B-
galactosidase activities were then measured. The Adjusted Luciferase Activity was
luciferase activity divided by B-galactosidase activity.

43

Identification of ERAE3 in LCC1 and LCC2 cells

To investigate the reason for the decreased ER activity in LCC2 cells, MCF-7, LCC1
and LCC2 cells were plated in IMEM/CSS for two days and then treated with E, 4-OH
TAM, or ICI. After forty-eight h, cells were harvested and cell lysate was analyzed by
western blotting using an antibody against an N-terminal epitope of wild-type ER (Figure
2.2). The regulation of ER expression in the three cell lines followed the same pattern; it
was up-regulated in the absence of E or presence of 4-OH TAM, and down-regulated in
the presence of E or ICI. Notably, an ER variant of approximately 61 kD was detected at
significant levels in LCC1 and LCC2 cells. In LCC2 cells, the protein level of the 61 kD
ER variant was comparable to that of the full length wild-type ER. The expression of the
61 kD ER variant was regulated by E, 4-OH TAM and ICI in the same way as that of
wild type ER.

The existence of the ER variant in LCC1 and LCC2 cells posed the interesting
question of whether the increase in its amount had anything to do with the phenotypes of
these cells, estrogen independence and tamoxifen resistance, respectively. To further
investigate the function of the ER variant in LCC1 and LCC2 cells, it was first
characterized molecularly. Based on the size of the protein, it was suspected to arise from
a deletion of exon 3. To test this hypothesis, a primer set was designed with the upstream
primer located in exon 2 and the downstream in exon 4. Total RNA was isolated from
MCF-7 and LCC2 cells, and RT-PCR was performed. In parallel, PCR reactions were
carried out with cDNA controls for both wild-type and A3 ERS (Figure 2.3A). In addition
to the 420 bp fragment predicted from wild type ER mRNA, a 320 bp fragment was

amplified from LCC2 mRNA. Both fragments were present in roughly equal amounts.

44

The 320bp fragment was then purified and sequenced. The sequencing results confirmed
the hypothesis that the 61 kD variant present in LCC1 and LCC2 cells was an ER exon 3
deletion, ERAE3 (Figure. 2.3B). One possible mechanism for the generation of the
ERAE3 mRNA found in LCC2 cells would be a splice site mutation in the genomic
DNA. In order to test for such a mutation, genomic PCR was carried out in MCF-7 and
LCC2 cells using a primer set with the first strand primer located in intron 2 and the
second in intron 4. The resulting 313 bp fragment was then purified and sequenced. No
genomic DNA mutations were observed within 122 bp upstream or 77 bp downstream of

the region encoding exon 3 in either MCF-7 or LCC2 cells (Data not Shown).

45

CSS E ICI TAM CSS E ICI TAM CSS E lCl TAM

 

 

_ 66kD

M M Adi"

Figure 2.2 Expression of ER in MCF-7 derivatives and identification of a 61 KD ER
variant in LCC2 cells. MCF-7, LCC1, and LCC2 cells were treated with E (10'9M), 4-OH
TAM (10‘6M), and ICI (10'9M) for 24 hours. Expression of ER was analyzed by Western

blotting using an ER antibody.

46

exon2 exon3 exon4
i —-bl r I ER

' ' 4— ' mRNA
1st 2nd

Marker H20 MCF-7 LC01 ER ERAE3

420 bp
320 bp

 

 

 

 

 

B
ERI micro] e l J
ml ~elcliol E 11

...TTCAAG GACATAA ATGAAAGGTG GGATAC...

Figure 2.3 Identification of ERA3 expression in LCC2 cells. A. Total mRNA from
MCF-7 and LCC2 cells were purified. RT-PCR using primers as indicated was performed
with ER and ER A3 cDNAs as controls. B. PCR products (420bp and 320bp fragments)
were purified and sequenced. The results were illustrated using a diagram.

Ectopic expression of ERAE3 in stably transfected MCF-7 and LC C 1 cells

The increased amount of ERAE3 in LCC1 and LCC2 cells led us to investigate
whether this variant might play a role in the phenotype of the two cell lines, estrogen
independence and tamoxifen resistance respectively. The ERA3 cDNA was inserted into
a pCDNA3 vector, and pCDNA3- ERAE3 was obtained. The ability of this construct to
express ERAE3 was confirmed by transient transfection into COS-7 cells followed by
western analysis of the cell lysate (Data not Shown). pCDNA3- ERAE3 or pCDNA3-
thRor were then transfected into MCF-7 and LCC1 cells, and stable transfectants were
selected in G418. Stably transfected cell clones expressing ERAE3 or wild type ER were
identified using RT-PCR and western blotting. Early passages of three independent
clones of MCF-7 and LCC1 expressing ERAE3 are Shown in Figure 2.4A. To determine
if these transfected MCF-7 and LCC1 cell lines maintained expression of ERAE3 in the

long term, they were cultured in the presence of G418 for more than 15 passages. Cells

were then harvested, and cell lysates were analde for ER expression by western
blotting (Figure 2.4B). While the transfected MCF-7 cells lost ERAE3 expression by
passage 10, the transfected LCC1 cells maintained expression of ERAE3 for at least 17
passages. This indicates that there is a difference in the ability of MCF-7 and LCC1 cells

to tolerate expression of ERAE3.

48

ERAE3 ERAE3
transfected transfected

 

 

MCF7 LCC1 LCCZ 2 6 7 28 44 126

ER
“.“r--fl-‘--ERA3

 

ERAE3 ERAE3

transfected transfected
M L L
C C CZ 6. 7 2 6, 7, 28.44126,
FCCPP.PPP.PPP.P.
712101010252525161717

ER

u-----.‘“"=- ERA3

Figure 2.4 A.Verification of ERAE3 expression in pCDNA3-ERAE3 transfected
MCF-7 and LCC1 cells. B. Loss of ERAE3 expression in stably transfected MCF-7
cells and sustained expression of ERAE3 in stably transfected LCC1 cells. Cells were
grown in IMEM/PBS. ERAE3 expression was analyzed by Western blotting using an
ER antibody. 2, 6, 7 were three independent pCDNA3-ERAE3 transfected MCF—7
cell clones. 28, 44, 126 were three independent pCDNA3-ERAE3transfected LCC1
cell clones. Their passages were indicated above.

49

Anchorage-independent proliferation of LCC1 cells expressing ERAE3

To investigate whether ectopic expression of ERAE3 would lead to tamoxifen
resistance in LCC1 cells, the ability of the stably-transfected cells to proliferate in the
presence of tamoxifen was examined. Control cells transfected with a wild-type ER
construct were also examined, as were untransfected MCF-7, LCC1, and LCC2 cells. The
expression of both ERAE3 and wild-type ER were confirmed by western blotting of cell
lysates (Figure 2.5A). Due to the fact that the phenotypes of MCF-7, LCC1, and LCC2
cells are not obvious on plastic, soft agar assays were used to assess the ability of the
transfectants to Show anchorage-independent proliferation. In these assays, cells were
plated in IMEM/CSS for two days, and then suspended in 0.3% agar containing
lxDMEM/F12 and 5% CSS with or without E or 4-OH TAM. The cells were then
incubated for 21 days, during which time they were fed every 5 days. The ability of cells
to form colonies in soft agar was interpreted by microscopy (Figure 2.58) and direct
counting of colonies (Figure 2.50).

As shown in Figure 2.5, MCF-7 cells only formed large colonies in the presence of E.
LCC1 cells formed colonies both with and without E, but not in the presence of 4-OH
TAM. LCC2 cells formed colonies in the absence of E and the presence of4-OH TAM.
The ability of these cells to form colonies in soft agar clearly correlated with their
reported phenotypes. The phenotype of LCC1 cells transfected with either pCDNA3-
thRor or pCDNA3- ERAE3 was unchanged; that is, they formed colonies in the absence
of E, but not in the presence of 4-OH TAM. These results indicate that ectopic expression

of ERAE3 is not sufficient to confer tamoxifen resistance to LCC1 cells.

50

Although MCF-7 cells were not able to sustain long-terrn expression of ERAE3, the
effect of ERAE3 expression on the anchorage-independent proliferation of early passage
transfectants was examined. The initial results of these soft agar assays suggested that
ectopic expression of ERAE3 abolished the ability of MCF-7 cells to proliferate in soft
agar in the presence of E (data not shown). However, this experiment could not be
repeated because expression of ERAE3 was not sustained during cell passage. However,
they suggest that the expression of ERAE3 had a negative impact on the proliferation of

MCF-7 cells, and therefore exerted a selective pressure towards its own elimination.

51

MCF—7 LCC1 LCC2 28 44 126 26 271 15

MCF-7 .

LCC1

LCCZ

LCC1/
ERA3

 

ICSS IE DTAM

w

Colony numbers
-h N

gusto b‘ofik
Vo°\9°”|_*_9_i"l"1i__i

pCDNA3-ERA3 pCDNA3 pCDNA3-ER

52

Figure. 2.5 A. Verification of ER and ERAE3 expression in stably transfected
LCC1 cells. Cells were grown in IMEM/PBS. Expression of ER and ERAE3 was
analyzed by Western blotting using an ER antibody. 28, 44, and 126 were three
independent pCDNA3-ERAE3 transfected LCC1 cell clones. 26 and 271 were two
independent pCDNA3-MEROL transfected LCC1 cell clones. 15 was pCDNA3
transfected LCC1 cell clone. B, C. Effect of ER and ERAE3 expressions on the
anchorage independent proliferation of LCC1 cells. LCC1/ERAE3 represents data
from Clone 28, which is similar to Clone 44 and Clone 126. LCC1/Eth represents
data from Clone 26, which is Similar to Clone 271. LCC1/pCDNA3 represents Clone
15’s data. Cells were plated in IMEM/CSS for two days and then grown in 0.3% soft
agar with or without E (10'9M) and 4-OH TAM (10'6M) for 21 days. Colonies larger
than 60 pm (more than 50 cells) were analyzed using microscopy and counted, and
six independent wells per treatment per sample were averaged. The results were

53

Discussion

In this study, we have identified the presence of an ER variant, ERAE3, in a series of
MCF-7 derivatives (LCC1 and LCC2 cells) that are estrogen independent and/or
tamoxifen resistant. Although there have been reports of the presence of ER variant
mRNAs in breast cancer cells, this is the first evidence that a Significant amount of an ER
variant is expressed at the protein level. Previously, the amount of ER variants in both
breast tumor tissues and tamoxifen resistant cell lines has been limited to a level that
could only be detected by sensitive techniques such as RT-PCR (10-18). This study
demonstrates that an ER variant can be present in amounts comparable to wild-type ER in
a tamoxifen resistant breast cancer cell line, i.e. LCC2 cells.

Previous studies have shown that a number of different ER variant mRNAs can co-
exist both in normal and transformed estrogen target cells (12). This suggests that the
existence of these ER variants generally does not result from mRNA splice Site
mutations. We have also shown here that the presence of the ERAE3 in LCC1 and LCC2
cells is not a result of a simple splice Site mutation. Our analysis does not, however, rule
out the complete loss of exon 3 and surrounding sequences from one allele of the ER
gene in LCC2 cells. Early evidence suggests that exon skipping is oneof the likely
causes of the presence of ER variant mRNAs in breast cancer cells (14), and our
observations are consistent with that suggestion. In addition, the increasing amount of
ERA3 from MCF-7 to LCC1 and LCC2 cells suggests that exon Skipping could
somewhat be regulated. How it is regulated and whether its regulation relates to estrogen

independence and/or tamoxifen resistance still need to be investigated.

54

A second interesting observation is that the increasing amount of ERAE3 from LCC1
to LCC2 cells correlates with decreased ERE responses to estrogen in these cells. ERA3
seems to act as a dominant negative ER in both LCC1 and LCC2 cells. This is consistent
with previous results(17, 21, 35). Furthermore, Since it cannot be maintained in stably
transfected MCF-7 cells, ERAE3 also seems to inhibit estrogen-dependent proliferation in
these cells. This observation is also consistent with earlier studies (21). In contrast,
ERAE3 expression can be maintained in LCC1 cells. This suggests that a change has
occurred in LCC1 cells that allows ERAE3 expression and it is possibly the same change
that confers estrogen independence. Such changes are likely to include genes that are
involved in estrogen regulation. Some possible candidates could be ER coregulators and
growth factors (24). The tolerance of ERAE3 expression in LCC1 cells further indicates
that ER function can be inhibited without interfering with proliferation at the same time.
Surprisingly, LCC1 cells are still sensitive to tamoxifen, suggesting that they are not
completely independent of ER function. A possible explanation could be that tamoxifen-
bound ER has unique effects on regulation of genes responsible for cell proliferation. It
could either suppress genes required for proliferation or activate genes that inhibit
proliferation.

The ectopic expression of ERAE3 alone does not confer tamoxifen resistance to either
MCF-7 or LCC1 cells. It suggests that ERAE3 alone cannot directly confer tamoxifen
resistance to estrogen-responsive breast cancer cells. This is consistent with the results of
other Similar studies (21). However, the possibility of an indirect or contributory effect of
increased ERAE3 expression on causing tamoxifen resistance cannot be excluded.

Perhaps the continued presence of ERAE3 imposes a selective pressure on cells (even in

55

LCC1 cells) to become less dependent of ER frmction. Therefore, over time, cells that are

tamoxifen resistant are favored.

56

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