"fibfifigi. 4 L.

. 3.6.5”

‘3;
afll»...

A .vau

nv‘
0......
“If.

3%? .5538’ g z ‘ gigag

a..\ .

.l.'

 

.1 , LIBRARIES
o 0 0 MICHIGAN STATE UNIVERSITY
EAST LANSING, MICH 48824-1048

This is to certify that the
dissertation entitled

THE ROLE OF MYC IN ANTIESTROGEN RESISTANCE OF
HUMAN BREAST CANCER CELLS

presented by

SHIBANI MUKHERJEE

has been accepted towards fulfillment
of the requirements for the

Doctoral degree in Microbiology and
Molecular Genetics

 

 

,1. CF‘.‘
/ K“ {7 \ \
>

 

/

Major Professor’s Signature
5? f/flr/ 26W;

Date

 

MSU is an Affirmative Action/Equal Opportunity Institution

-<-_t--.-.-.-----u-n-v-o-Du-~—

-.-.-.- -Ohlw“

PLACE IN RETURN BOX to remove this checkout from your record.
To AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

_._J‘
2/05 mmmms

THE ROLE OF MYC IN ANT IESTROGEN RESISTANCE OF
HUMAN BREAST CANCER CELLS

By

Shibani Mukherj ee

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Microbiology and Molecular Genetics

2005

ABSTRACT

THE ROLE OF MYC IN ANTIESTROGEN RESISTANCE OF
HUMAN BREAST CANCER CELLS

By

Shibani Mukherjee

Antiestrogens are among the most effective and least toxic of systemic breast
cancer therapies. However, acquired antiestrogen resistance is a major cause of treatment
failure. Understanding the molecular mechanisms underlying the proliferative effects of
estrogen in breast cancer cells is critical for determining how breast tumors respond to
but eventually often become resistant to antiestrogen therapy. Estrogen rapidly induces
the expression of the transcription factor c-Myc, which is required for estrogen-
stimulated proliferation of breast cancer cells. c-Myc levels are decreased in the presence
of antiestrogen, and deregulated c-Myc expression has been implicated in antiestrogen
resistance. We investigated the mechanism(s) by which c-Myc mediates estrogen-
stimulated proliferation and contributes to cell cycle progression in the presence of
antiestrogen.

The MCF-7 cell line, which is a model of estrogen-stimulated, antiestrogen-
sensitive human breast cancer, was used in our studies. First, the regulation of cell cycle
progression and c-Myc expression by estrogen and antiestrogen was confirmed in the
MCF-7 cell line. Next, we established stable MCF-7 derivatives with inducible c-Myc
expression, and confirmed that ectopic c-Myc expression is sufficient to induce cell cycle

progression in the presence of antiestrogen. We then observed that in antiestrogen-

treated cells the elevated mRNA and protein levels of pZIWAWCIPI, a cell cycle inhibitor,
decreased upon either c-Myc induction or estrogen treatment. Using RNA interference to
suppress c-Myc expression, we demonstrated that c-Myc was required for estrogen-
mediated decreases in pZIWAFl/Cm. In addition, expression of pZIWAFI/Cm suppressed
the ability of c-Myc to overcome an antiestrogen arrest. This suggested that the decrease

in p21WAFl/CIP1

was necessary for c-Myc-mediated cell cycle progression in the presence
of antiestrogen. Previous work has shown that decreasing p21w’m/CIPl using antisense
oligonucleotides is sufficient to induce cell cycle progression in the presence of
antiestrogens. Together with our data, this suggests that a key mechanism by which c-
Myc promotes proliferation of breast cancer cells in the presence of antiestrogens is by
decreasing p21 levels. Finally, we studied a MCF—7 derivative that serves as a model for
acquired antiestrogen resistance. Neither c-Myc nor pZIWAF"CIPl was regulated by
estrogen or antiestrogen in this cell line. The p21‘mwcm1 levels in the antiestrogen-
resistant cells increased when c-Myc expression was suppressed, indicating that the loss
of pZIWAF new regulation was a consequence of constitutive c-Myc expression.

In summary, these studies identify pZIWAFUCIPl as an important target of c-Myc in
breast cancer cells, and provide a link between estrogen, c-Myc and the cell cycle
machinery. They further suggest that aberrant c-Myc expression, which is frequently
observed in human breast cancers, can contribute to antiestrogen resistance by altering

IWAFl/CIPI

p2 regulation.

To Dadu

iv

ACKNOWLEDGEMENTS

I would like to thank my mentor Dr. Conrad for giving me the opportunity to do
research that has excited and inspired me throughout these years. I am grateful for her
excellent mentoring, she kept me focused and gave me advice and encouragement when
research was tough, and then rejoiced with me and challenged me to do even better when
things were going well. I would like to thank Dr. Esselman for always taking the time to
meet with me, make me smile and then address all my concerns. I am gratefiil to Dr.
Fluck whose energy and tireless enthusiasm for science have been a source of inspiration
for me, and to Dr. McCabe for sharing with me her passion for research and the secrets to
a high “batting average”. I am also thankful to Dr. Miksicek; his insightful comments
and questions have always motivated me to think harder about my work. I am indebted
to Dr. Schwartz, for his guidance, and because he gave an inexperienced student a chance
at a wonderful graduate experience.

A big thanks to all previous and current members of the Conrad lab, Andy
Skildum, Feng Han, Yuping Tang, Rami Saba, Emily Flynn, Yi Zheng, and Sarah Santos;
my work would not have been possible without their help and friendship. I would like to
especially thank Hemant Varma for his encouragement over the years. I am also grateful
to all my other fi’iends, particularly Gauri J awdekar, Padmesh Venkitasubramanian,
Weizhong Wang, and Trevor Wagner, for their support. I am humbled by the generosity
of Robin Garber and Nan Wagner, who have provided me with a home away from home.
Finally, I would like to thank my extended family in India, my parents, and my sister

Nandini, who despite being far away, have stood by me every step of the way.

TABLE OF CONTENTS

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

Literature Review ................................................................................ 1
Breast Cancer Incidence, Mortality and Risk Factors ....................................... 2
Estrogen and Breast Cancer ..................................................................... 3
Antiestro gen Therapy ............................................................................. 5
Antiestro gen Resistance ........................................................................ 9
Estrogen Receptor: Mechanism of Action of Estrogens and Antiestrogens. . . . . . . . .....10
Eukaryotic Cell Cycle .......................................................................... 14
Regulation of G1 to S Phase Transition by Cyclin-Dependent Kinases ................. 16
Estrogen and Cell Cycle Regulation .......................................................... 20
The Protoonco gene c-myc ...................................................................... 21
c-Myc and Cell Cycle Regulation ............................................................. 25
Estrogen and c-Myc .............................................................................. 27
Mechanisms of Antiestrogen Resistance ...................................................... 28
References ......................................................................................... 30
CHAPTER 2

Regulation of Cell Cycle Progression by Estrogen, Antiestrogen and c-Myc Induction in
MCF-7 cells

Abstract .......................................................................................... 46
Introduction ...................................................................................... 47
Materials and Methods ......................................................................... 49
Results ............................................................................................ 53
Discussion ........................................................................................ 68
References ........................................................................................ 73
CHAPTER 3

c-Myc Suppresses pZIWAFUCIP‘ Expression During Estrogen Signaling and Antiestrogen
Resistance in Human Breast Cancer Cells

Abstract ........................................................................................... 80
Introduction ...................................................................................... 81
Materials and Methods ......................................................................... 83
Results ............................................................................................ 88
Discussion ....................................................................................... 104
References ....................................................................................... 1 12

vi

LIST OF FIGURES

CHAPTER 1

Figure 1: Schematic representation of the domain organization of ERa. . . . . . . . . . . ....11
Figure 2: Molecular mechanisms underlying ER signaling .............................. 13
Figure 3: Model of the eukaryotic cell cycle .............................................. 15
Figure 4: Simplified model of the cyclin/CDK pathway ................................. 17
Figure 5: Schematic representation of the c-myc gene organization ................... 23

and domain structure of the c-Myc protein.

CHAPTER 2

Figure 1: Cell cycle progression is regulated by ICI and E2 in MCF-7 cells ......... 54

Figure 2: Regulation of c-Myc by E2 and ICI in MCF-7 cells .......................... 57

Figure 3: Effect of c-myc siRNA on c-Myc expression and cell cycle ................. 59
progression in E2-treated MCF-7 cells.

Figure 4: Construction of MCF-7/Myc stably transfected cell lines ......................... 61

Figure 5: Characterization of cell lines with inducible c-Myc expression ............ 64

Figure 6: Induction of ectopic c-Myc and cell cycle kinetics in ICI-treated ......... 67
MCF-7 cells.

CHAPTER 3

Figure 1: Effects of c-Myc induction on cell cycle progression and protein ........... 90
expression in ICI-treated cells, and in cells infected with an adenovirus
expressing p21.

Figure 2: Analysis of p21 protein and mRNA levels, and p21 promoter-luciferase..95
activity following c-Myc induction in ICI-treated cells.

Figure 3: Effect of c-myc siRNA on p21 expression in E2-treated MCF-7 cells. . ....97

vii

Figure 4: Analysis of c-Myc expression in an antiestrogen-resistant cell line. . . . . ......99
Figure 5: Analysis of p21 protein and mRNA levels in LCC9 cells .................... 101
Figure 6: Effect of c-myc siRNA on p21 expression in LCC9 cells ..................... 103

Figure 7: Proposed model for the role of c-Myc in E2 signaling and antiestrogen. . . 107
resistance.

viii

CHAPTER 1

LITERATURE REVIEW

LITERATURE REVIEW

Breast Cancer Incidence, Mortality and Risk Factors

Worldwide, more than a million women are diagnosed with breast cancer every
year, accounting for a tenth of all new cancers and 23 percent of all cancer cases in
women. Breast cancer is the most common cancer among women in industrialized
countries and second only to cervical cancer in developing countries. Last year, over
275,000 new cases of breast cancer were diagnosed in the United States, and more than
7000 of those were in Michigan. The incidence of breast cancer has increased, but the
number of deaths due to breast cancer seems to be declining. Breast cancer still remains
the second leading cause of cancer mortality, with more than 500,000 deaths worldwide
and over 43,000 deaths in the United States each year (1, 2).

The National Cancer Institute estimates that currently one in seven women in the
United States will develop breast cancer during her lifetime. In the 19708, the lifetime
risk of being diagnosed with breast cancer was one in ten women and since then has risen
gradually. This increase may be explained by a variety of factors. It is believed that the
rise is partly due to better detection tools, and the fact that women are living to an older
age, when their risk increases (3). It is also possible that lifestyle changes over the years
may have increased the chance of developing breast cancer. There are several factors
that are known to increase a woman’s chance of developing breast cancer. These factors
include older age, early onset of menarche, a late pregnancy or no pregnancy, a family
history of breast cancer, and a previous breast biopsy (whether positive or negative) (4-

6). Other factors such as age at menopause, dense breast tissue on a mammogram, use of

birth control pills or hormone replacement therapy, high-fat diet, alcohol, physical
activity, obesity, and environmental exposures are also thought to influence a woman’s
risk, but their exact contributions to increased risk remains to be determined. Most of
these risk factors are obviously associated with being a female. Breast cancer can occur
in men; this year approximately 1700 new cases will be diagnosed and about 460 men
will die from breast cancer in the United States. However, this accounts for less than 1%
of all breast cancer cases (7). Several risk determinants, such as onset of menarche,
pregnancy, hormone replacement therapy and birth control pills, relate to changes in the
levels of hormones such as estrogens. Exactly how estrogens affect breast cancer risk
remains controversial, and it is thought to greatly depend on the timing and duration of
the exposure. Estrogenic stimuli during the postmenopausal years are associated with a
higher breast cancer risk, but this may not be true of premenopausal exposure (8).
Estrogens can be considered to act as either tumor promoters (agents that stimulate
proliferation/survival of existing transformed cells) or as tumor initiators (factors that

induce the genetic damage that leads to cellular transformation).

Estrogen and Breast Cancer

Estrogens are cholesterol derivatives that regulate the development,
differentiation and proliferation of reproductive tissues. They also exert effects on the
bone, liver and cardiovascular systems. The major estrogenic compounds secreted by the
ovaries are estrone, estriol and l7B-estradiol, of which the latter is the most potent
estrogen. Their levels are elevated significantly during puberty, fluctuate at these

increased levels during the menstrual cycle, and finally decrease again at menopause.

The main site of estrogen synthesis in postmenopausal women changes from the ovaries
to the peripheral tissues. The adrenal glands make androstenedione, and the adipose
tissue that is a major site of aromatase activity converts the androstenedione into
estrogens (9).

Hormones have had a suspected role in breast cancer etiology for over a century,
and exposure to endogenous and/or exogenous estrogens is now accepted as a factor that
can influence susceptibility to breast cancer (10). In 2003, the National Toxicology
Program listed steroidal estrogens as carcinogens for the first time. The role of estro gens
in breast cancer has primarily been attributed to their mitogenic activity in established
tumors rather than to tumor initiation (11). However, it has been proposed that estrogen
metabolites can directly cause DNA damage, and may be involved in tumor initiation.
Human breast tissue can be a site of oxidative metabolism of estrogens due to the
presence of specific cytochrome P450 enzymes. There is evidence suggesting a role for
the oxidative metabolites of estrogens, in particular the catechol estrogens, in
carcinogenesis. In addition, upon further oxidation the catechols can give rise to reactive
quinones that are capable of forming direct adducts in DNA, and redox cycling to
generate reactive oxygen species that can cause oxidative damage. Such events could
define reactive estrogen metabolites as initiators, rather than simply promoters of
carcinogenesis (12, 13).

Although the actual role of estrogen metabolites in initiating tumors in viva
remains controversial, the mitogenic effect of estrogens on estrogen-responsive breast
tumors has been known for a long time. In 1896, Sir George Beatson provided the first

insight into the regulation of breast cancer proliferation by estrogens when he observed

that surgical removal of ovaries in premenopausal women with advanced breast cancer
led to remarkable tumor regression. Subsequently, many studies identified estrogens as
key ovarian hormones involved in both normal and cancerous proliferation of the breast
( 14, 15). Ovariectomy in prepubertal mice prevents mammary gland development and
this can be reversed upon estrogen supplementation, indicating estrogen’s role in normal
breast development and proliferation. Studies using athymic nude mice xenografted with
human breast cancer cells have demonstrated that tumor formation and growth require the
presence of estrogens (16—18). The role of estrogens in breast tumor growth is now well
established, and estrogen-stimulated proliferation of breast cancer cells is described in
detail in subsequent sections. A majority of primary human breast cancers express the
estrogen receptor (ER), and approximately 40% require estrogenic steroids for
proliferation (19). The key role for estrogen in breast cancer proliferation has provided
the rationale for the development of antiestrogenic compounds for the treatment of breast

cancers.

Antiestrogen Therapy

Currently, primary therapies for breast cancer include surgery, cytotoxic
chemotherapy and radiation therapy. Following primary therapy, depending upon
receptor and menopausal status of the patient, various endocrine manipulations can be
used for systemic adjuvant therapy. The general principle of endocrine therapy is to
inhibit the mitogenic effect of estrogens. Unlike chemotherapy, which is also a systemic

treatment, endocrine therapy is well tolerated and has relatively few side effects.

Cytotoxic chemotherapeutic agents lack selectivity for tumor cells and target all
rapidly growing cells, including tumor cells, hematopoietic cells, cells lining the digestive
tract and hair follicle cells. Common side effects of chemotherapy therefore include
immunosuppression, gastrointestinal disturbances, and alopecia. Less tolerable problems
associated with certain drugs include neurotoxicity, renal failure, and cardiotoxicity, and
the mutagenic potential of certain agents can cause the development of rare complications
such as leukemia (20). Unlike chemotherapy, endocrine therapy primarily blocks
proliferation in breast tissue. However, depending upon the form of endocrine therapy
used, uterine, cardiovascular and skeletal tissues can also be affected. Three rare but life
threatening side effects associated with endocrine therapy include increased risk of
endometrial cancer (cancer of the uterine lining), deep vein thrombosis (blood clots in
major veins) and pulmonary embolism (blot clots in the lung). Some forms of endocrine
therapy are associated with several positive side effects, including protective effects
against osteoporosis and potentially cardiovascular diseases (21-23).

Early endocrine manipulations involved ovarian ablation using surgical
oophorectomy or ovarian irradiation. Chemical forms of reversible ovarian ablation are
still used in the endocrine therapy of some premenopausal patients (24). In the mid-
19503, compounds that could act as estrogen antagonists and chemically block effects of
estrogens were first developed and used as fertility agents. Their ability to induce
responses in some breast cancer patients quickly became apparent; however they induced
significant toxicity (25). In the early 1970s, the first studies were carried out using a new
antiestrogen called Tamoxifen (Nolvadex), which was found to have minor side effects

(26). Tamoxifen has subsequently become the most frequently prescribed antiestrogen,

and total exposure to Tamoxifen has reached over 10 million patient years. Worldwide
clinical trials indicate that the five and ten year mortality rates of breast cancer patients
can be reduced 20-25% by Tamoxifen treatment (21, 22).

Antiestrogens are now the most widely administered endocrine agents for the
management of ER-positive breast cancers, and act primarily by competing with
estrogens for binding to the ER. There are two broad classes of antiestrogens,
nonsteroidal or partial antiestrogens such as Tamoxifen and Raloxifene, and steroidal
antiestrogens like ICI 182,780 (ICI, Faslodex) that are “pure” estrogen antagonists (27).
This nomenclature reflects the fact the partial antagonists such as Tamoxifen and
Raloxifene inhibit the action of the ER, but also display some estrogenic characteristics.
For example, while both Tamoxifen and Raloxifene inhibit estrogen-stimulated growth in
breast tissue, they exhibit estrogenic activity in bone and cardiovascular tissue
(Tamoxifen and Raloxifene) and uterine tissue (Tamoxifen). Due to the fact that these
compounds can act as both estrogen agonists and antagonists, they are also called
_S_elective Estrogen Receptor Modulators (SERMs) (28).

Hormonal therapy has advanced rapidly in the last decade. Tamoxifen still
benefits many women, but it is no longer the only option available (29, 30). The steroidal
estrogen antagonist, ICI, has a 100 fold higher binding affinity than Tamoxifen, and has
no known agonist activities. In addition to blocking the binding of B2 to the ER, ICI
causes increased receptor degradation, decreases receptor dimerization, and disrupts ER
nuclear localization. Due to the fact that pure antiestrogens decrease the amount of ER
available for activation, they are increasingly referred to as Estrogen Receptor

Qownregulators (ERDs). Since ICI decreases the amount of ER available it also prevents

ER activation via alternative pathways through other mitogens and grth factors, and
thus maybe more effective than the SERMs (27, 31). In 2002, the US. Food and Drug
Administration (FDA) approved the use of ICI for treatment of ER-positive metastatic
breast cancer in postmenopausal women who had relapsed on Tamoxifen.

Another approach to endocrine therapy involves the use of compounds called
aromatase inhibitors (32). This method contrasts with antiestrogen treatment in that
instead of competing with estrogens for binding to the ER, these compounds reduce the
amount of estrogen being produced by the body. As discussed previously, in
postmenopausal women, the main source for estrogens is the conversion of
androstenedione into estrogens via aromatase enzymes (9). Aromatase inhibitors, such as
Anastrazole, Letrozole and Exemestane, block the action of the aromatase enzyme and
thereby reduce the levels of estrogens in postmenopausal patients. In 2002, the FDA
approved Anastrazole for adjuvant therapy in postmenopausal patients with early stage
ER-positive breast cancer, and in late 2004, clinical trials concluded that Anastrazole
should be the preferred initial treatment in this subgroup. The trials indicated that it is
more effective than Tamoxifen in preventing recurrence as well as occurrence in the
other breast, and has fewer side effects. However, aromatase inhibitors increase the risk
of osteoporosis. Importantly, they are not effective in premenopausal women since their
estrogen production is still primarily ovarian, and the ovaries can overcome an estrogen
blockade by increasing the levels of luteinizing hormone and follicle stimulating
hormone. In contrast, SERMs decrease osteoporosis, and can be administered

irrespective of menopausal status (33, 34).

Antiestrogen Resistance

Future clinical trials will help detemrine what the order of endocrine treatments
should be, and as better SERMs, ERDs and aromatase inhibitors are developed, the
number of treatment options is increasing. This is of significance given that many
patients, who have initially antiestrogen-sensitive tumors, and a majority of those with
metastatic disease, eventually relapse with the development of acquired resistance to
antiestrogens (35). While it is well known that tumors develop resistance to Tamoxifen
during or after the five year recommended course of treatment, more recently there is also
evidence for the development of resistance to Anastrazole and ICI. Patients who develop
resistance to one form of endocrine therapy often still respond to other endocrine
treatments (36, 37). Therefore, increasing the number of treatment options may provide
the opportunity to extend the sequence of endocrine regimens before cytotoxic
chemotherapy is required. As the prognosis for patients with such tumors is generally
poor, the development of resistance to Tamoxifen and other hormonal therapy represents
a major obstacle in the treatment of breast cancer patients. This may have even greater
significance now that there is more widespread use of Tamoxifen, which in 1998 was
approved by the FDA for breast cancer chemoprevention (38, 39).

Eventually, a patient may develop resistance to all forms of endocrine therapy,
and a more permanent solution therefore lies in determining the mechanism by which
resistance arises. The molecular changes underlying the progression of breast cancer
cells from an estrogen-dependent, antiestrogen-sensitive to an estrogen-independent,
antiestrogen-resistant phenotype are poorly characterized. Understanding the

mechanisms by which estrogens and antiestrogens regulate proliferation of breast cancer

cells is crucial, as it would provide insight into how antiestrogen resistance may arise and
suggest strategies to prevent or reverse its development. The following sections review
what is currently known about the molecular targets through which estrogens and
antiestrogens regulate cell cycle progression and thus growth of breast tumors, and about

potential mechanisms whereby antiestrogen resistance may arise.

Estrogen Receptor: Mechanism of Action of Estrogens and Antiestrogens

Estrogens mediate their effects by binding to a steroid hormone receptor protein
called the ER. There are two isoforms of ER, the well-characterized ERa and the more
recently described ERB (40). Certain functional domains of the ERoc and ERB exhibit a
high degree of homology, whereas considerable divergence is apparent in the N terminus.
Both receptors have a similar binding affinity for 17-13 estradiol (E2), however they differ
in tissue expression and play different roles depending on cell type and promoter context
(41). Based on studies in knockout mice, ERa is thought to be involved in breast
development and proliferation, while ERB is not (42). i Importantly, ERa is present in
most estrogen-dependent breast cancers and the role of ERoc in breast tissue and cancer is
well established, whereas the role of ERB is less clear. Therefore, the following sections
will focus on estrogen signaling mediated through ERor, and henceforth unless otherwise
noted ER refers to ERa.

The ER is a transcription factor comprised of a DNA binding domain, a ligand
binding domain and two transcriptional activation domains, one at the N-terminus (AF 1)
and the other at the C-terminus (AF 2) (Figure l) (43, 44). The molecular mechanisms

underlying ER signaling are complex, and at least four distinct pathways have been

10

1 1 85 250 311 551 595

 

 

 

 

 

 

 

 

 

 

Nl-l2 a N3 c o E F - COOH
L v 1 _ _ngf—J ¥ v J
Transactivatlon DNA Hinge Ligand binding

(AF 1) binding Dimerization
Dimerization Transactivation (AF2)

Figure 1: Schematic representation of the domain organization of ERa. (adapted
from Ruff M. et a1. Breast Cancer Res. 2000; 2(5): 353-359 Figure 1). The ER was first
divided into six regions (A-F), based upon sequence homology between human ER and
chicken ER, with A, B and C being the most highly conserved regions. Subsequently ER
from other species (mouse, rat, Xenopus and trout) was sequenced, and comparisons of
amino acid sequences demonstrated that the basic structural domains have been very
highly conserved throughout evolution. These include an amino terminal transactivation
domain (AF 1), a DNA binding domain, a hinge region, and a carboxyl terminal ligand
binding domain, which also contains the second transcriptional activation domain (AF 2).

ll

defined (Figure 2) (45). In the classical ligand-dependent mechanism of ER activation,
E2-bound ER dirnerizes and activates transcription by binding to a specific DNA
sequence called an estrogen response element (ERE), which is found in the
promoter/enhancer regions of many E2-regulated genes. The C-terminal AF 2 domain is
involved in this ligand induced ER activation (46-48). In the second, ligand-dependent
but ERE-independent mechanism, E2-bound ER alters transcription of genes through
protein-protein interactions with other transcription factors, such as APl (F os/Jun) or
SP1, which tether the activated ER to DNA (49, 50). The third pathway involves a
ligand-independent mechanism, wherein intracellular kinase pathways are activated, for
example by growth factors such as EGF or IGF-l, leading to phosphorylation of the AF 1
domain and thus the activation of ER (51). Finally, there is increasing evidence for a
mechanism involving nongenomic signaling of the ER. In this mechanism, E2 activates a
subpopulation of ER outside the nucleus, resulting in activation of intracellular signal
transduction pathways such as the Shc pathway and Src/Erk phosphorylation cascade,
which generate rapid tissue responses (52-56).

Ultimately, various coactivators and corepressors mediate ER transcriptional
activity. The ER conformation varies based upon the ligand that is bound to it, and this
results in the recruitment of different cofactors to the promoters. Upon E2 binding to the
ER, a conformational change is induced in the AF 2 domain that results in the recruitment
of coactivators, such as members of the p160 family that includes SRC-l, AIBl and
GRIPl. This in turn leads to chromatin remodeling through histone modifications, which
then facilitates RNA polymerase H transcription of target genes. The antiestrogens,

Tamoxifen and ICI, both inhibit recruitment of coactivators to the ER in breast cancer

12

 

@ q 3. Ligand-independent
_> (9%
K.» R ®

 

 

 

ERE

E2 §____> 1. Classical
\_> E2 E3? ERE

Qflsfidepe
pendent
g \ \ A

4- Nongenomlc L Rapid tissue responses J

 

 

 

 

 

 

 

 

 

 

 

 

Figure 2: Molecular mechanisms underlying ER signaling (adapted from Hall J.M. et
al. J Biol Chem. 2001; 276(40): 36869-72 Figure l). The molecular mechanisms
underlying ER signaling are complex, and at least four distinct pathways have been
defined:

1) Classical mechanism - E2-bound ER dimerizes, and activates transcription upon
binding to the estrogen response element (ERE).

2) ERE-independent mechanism - E2-bound ER associates with DNA-bound
transcription factors and thereby regulates the transcription of genes containing
alternative response elements.

3) Ligand-independent mechanism - Growth factors (GF) bind to growth factor
receptors (GFR) and activate intracellular kinase pathways, leading to phosphorylation
(P) and activation of ER.

4) Nongenomic mechanism - E2 activates a population of ER that lies outside the
nucleus, and is linked to signal transduction pathways that generate rapid tissue
responses.

13

cells, and Tamoxifen-liganded ER has been shown to recruit the corepressors, NCoR and
SMRT, which negatively regulate transcription. Tamoxifen can have partial agonist
activity, and in some cell/promoter-specific contexts, Tamoxifen-bound ER is able to
recruit coactivators and activate transcription. In contrast, ICI has no known agonist
activity, and instead induces ER degradation via the ubiquitin/proteasome pathway (57-
61).

There is increasing evidence that altered levels of at least some coregulators can
contribute to deregulation of ER-mediated gene expression and proliferation in the
presence of antiestrogens. For example, SRC-l knockout mice are partially resistant to
the effects of E2, and show decreased mammary gland proliferation (62). Another SRC
family member called AIBl is overexpressed in 60% of primary breast tumors, and
amplified in 10% of breast tumors, and can promote estrogen-independent proliferation
of breast cancer cell lines (63, 64). Notably, patients who receive Tamoxifen therapy and
have high levels of AIBl expression have a shorter period of disease free survival, which
is indicative of the development of Tamoxifen resistance (65). The role of altered
receptor coactivator or corepressor expression in antiestrogen resistance is under intense
investigation; however the contribution of most coregulators remains unclear, and

understanding their roles is confounded by their firnctional redundancy.

Eukaryotic Cell Cycle
Mitogens, including estrogens, impinge on the cell cycle machinery and increase
proliferation. Cell division in most somatic cells is an ordered process with multiple

levels of regulation (66). Each round of division or cell cycle is divided into four phases

l4

4n

 
 
   

<2 Estrogen

<:l pRB

> 2n < 4n phosphorylation

Figure 3: Model of the eukaryotic cell cycle. Cells replicate their DNA (from 2n to
4n) and undergo cell division through a highly ordered sequence of events called the cell
cycle. Each round of cell division/cell cycle is composed of four distinct phases: the
DNA synthesis (S) phase, the Mitosis (M) phase and two gap phases (G1 and G2), during
which signals from the intracellular and extracellular environments are assessed to
determine whether conditions are appropriate for progression into the next phase. Cells
in the G1 phase are stimulated by mitogens, such as E2, which enhance progression
through this phase by activating the cyclin-dependent kinases (CDKs). CDK4/6 function
in early G1 and are activated by binding to D-type cyclins, while CDK2 acts in
conjunction with cyclins E/A and is necessary for progression through late G1 and entry
into S phase. These kinases phosphorylate and inactivate the pRB family of negative cell
cycle regulators during mid to late G1 phase. This results in the release of transcription
factors that regulate the transcription of genes that are important for passage from G1 to S
phase.

(Figure 3) (67-69). During the S phase, the cell duplicates its chromosome, and during
the M phase the chromosomes segregate and the cell membrane invaginates to complete
cytokinesis. The S and M phases are preceded by two phases called Gapl (G1) and Gap2
(G2) respectively. During these phases, signals from the intracellular and extracellular
environments are assessed to determine whether conditions are appropriate for
progression into the next phase (70, 71). Cells arrest in the G1 phase under unfavorable
conditions until conditions improve, and differentiated cells may exit the cell cycle
altogether at G1 and enter G0, which is a quiescent state. Cells must respond to
numerous extracellular growth factors, mito gen antagonists, differentiation inducers and
spatial cues in exercising their commitment to exit the G1 phase and enter S phase. Cells
are sensitive to these stimuli until they reach a point late in G1 called the restriction point,
after which they can complete their cell division cycle if supplied with factors that
support their viability (72). Malignant cells characteristically show a lack of

differentiation and of appropriate cell cycle regulation (73).

Regulation of G1 to S Phase Transition by Cyclin-Dependent Kinases
Cyclin-dependent kinases (CDKs) are serine-threonine kinases that control the G1
to S phase transition. Their activity in turn is tightly regulated by multiple mechanisms,
including activation by specific complex formation with regulatory cyclins, inhibition by
binding of CDK inhibitors, and specific phosphorylation/ dephosphorylation events
carried out by CDK-activating kinase (CAK) and CDC25A phosphatase (Figure 4) (74).
The catalytic activity of CDK5 requires binding of a cyclin protein at a one to one

stoichoimetry. Cyclins are proteins with short half lives and their levels are tightly

16

p21
coczsA\ 927 CAK

 

 

 

L
6 @ ....
®®
—) S phase
m entry
'1'
CDC25A p21 CAK
p27

Figure 4: Simplified model of the cyclin/CDK pathway (adapted from Sherr C.J.,
Science, 1996; 274(5293): 1672-1677 Figure 2). CDK activity is regulated by multiple
mechanisms, including activation by regulatory cyclins, inhibition by binding of CDK
inhibitors, and specific phosphorylation/ dephosphorylation events carried out by CDK-
activating kinase (CAK) and CDC25A phosphatase. In this model, cyclinDl/CDK4
complexes phosphorylate pRB family members, thereby releasing the E2F family of
transcription factors. E2F release results in the transcription of genes required for the S
phase, and in the transcription of cyclin B and cyclin A, which then activate CDK2. In a
positive feed back loop CDK2 complexes can also phosphorylate pRB and thus amplify
the proliferative signal of the cyclin-CDK pathway.

controlled by mitogenic stimuli and cell cycle phase (75, 76). By modulating the levels
of specific cyclins, mitogens such as E2 can regulate the activities of specific CDKs
during the different phases of the cell cycle. CDK4 and the related CDK6 act early in the
G1 phase and bind to D-type cyclins, which include cyclin D1, D2 and D3. In the
majority of cell types, including most human breast cancer cells, cyclin D1 is considered
the main regulatory subunit of CDK4. CDK2 functions in conjunction with cyclins E or
A and is necessary for progression through late G1 and entry into S phase (77, 78).
Cyclin-CDK activity is also controlled by two families of CDK inhibitors: the
INK4 family that includes p16‘NW (p16), and the pZIWAFl/Cm (p21) /p27"“’1 (p27)
family (79). The p16 protein was initially discovered as a tumor suppressor, and is a
specific inhibitor of CDK4/6. It binds to and inactivates these kinases by preventing
formation of cyclin D-containing complexes (80). In contrast to p16, both p21 and p27
bind to cyclin-CDK complexes and not CDKs alone, and they can bind to and inhibit
activation of both cyclin-CDK4/6 and cyclin-CDK2 complexes (81-83). Under certain
experimental conditions, p21 and p27 may also enhance the formation of cyclin D-CDK4
complexes by serving as assembly factors (84-86). The stoichoirnetry of CDK inhibitor
binding to the cyclin-CDK complexes may detemrine whether they function as inhibitors
or activators, however this remains controversial. CDK inhibitor function may also be
modulated by other mechanisms such as proteolysis or phosphorylation (87, 88).
Another level of regulation is based on the cellular concentrations of certain cyclin-CDK
complexes. Increasing concentrations of cyclin Dl-CDK4/6 complexes can provide
additional binding sites for p21 and/or p27 and titrate these inhibitors away from CDK2

complexes, resulting in CDK2 activation (89, 90).

18

An additional level of CDK regulation is provided by CAK and CDC25A
phosphatase (74). CAK phosphorylates a conserved threoninc residue on the CDK3, and
phosphorylation at this site is required for CDK activation. CDK activation also involves
removal of inhibitory phosphates by the CDC25A phosphatase. CDC25A belongs to a
family of dual-specificity phosphatases that remove the inhibitory phosphates threoninc-
14 and tyrosine-15 on the CDKs (91, 92). CDC25A activity in turn can be increased
upon phosphorylation by cyclinE-CDKZ, suggesting that a positive feedback loop exits,
wherein cyclin E-CDKZ phosphorylates and activates CDC25A that then activates CDK2
complexes by dephosphorylation (93). Both CDK inhibitor families are known to block
the activating phosphorylation of CDK by CAKs (94). In addition, CDC25A and p21
have been shown to compete for the same binding site on cyclin-CDK2 complexes. p21
inhibits CDC25A association with cyclin-CDK2 and thus blocks dephosphorylation of
CDK2. Conversely, CDC25A associates with cyclin-CDK2 and protects it from
inhibition by p21 (95).

The activated cyclin-CDK complexes ultimately phosphorylate a family of pocket
proteins, pRB, p107 and p130, which are negative regulators of proliferation (96).
Hypophosphorylated pocket proteins bind to and repress the E2F/DP family of
transcription factors that regulate genes important for passage from G1 to S phase (97,
98). In addition, EZF-bound pRB complexes recruit chromatin modifying enzymes to
actively repress transcription of these genes (99). Phosphorylation of the pocket proteins
leads to the release of E2F/DP proteins and results in the transcription of genes required
for the S phase, such as dihydrofolate reductase, thyrnidylate synthase, thymidine kinase

and DNA polymerase a (73, 100, 101). Activation of E2F-l also leads to transcription of

19

cyclin B and A that in turn activate CDK2. Activated CDK2 then targets substrates
which play a role in DNA replication; these include the origin-recognition complex
proteins (ORCs), minichromosome maintenance proteins (MCMs), and CDC6, all of
which assemble into preinitiation complexes. CDK2 also further phosphorylates pRB,
and reinforces the initial activation of E2Fs, and thus amplifies the proliferative signal of

the cyclin-CDK pathway (73).

Estrogen and Cell Cycle Regulation

E2 stimulates resting (GO/G1 phase) cells to enter the cell cycle and enhances
progression through the G1 to S phase of the cell cycle (Figure 3) (102). As described
above, E2 mediates its mitogenic effects by activating the ER and acting directly and/or
through signaling pathways, to induce the expression of genes required for cell
proliferation, and repress genes that inhibit proliferation (Figure 4). Antiestrogens cause
a GO/Gl arrest by binding the ER and preventing the activation of genes by E2 (103,
104). Over the last two decades significant progress has been made in understanding the
A cellular components involved in cell cycle progression, and cyclin-dependent kinases
(CDKs) have been identified as key mediators of E2-stimulated proliferation.

Upon E2 treatment of E2-starved or antieStrogen—treated cells, an increase in
CDK4 and CDK2 activities is observed, accompanied by increases in pRB
phosphorylation (105, 106). Treatment with E2 leads to an early increase in the levels of
cyclin D1, to decreases in the levels of CDK inhibitors, and to activation of CDC25A
(107). Cyclin D1 binds to CDK4, resulting in an increase in CDK4 activity, pRB

hyperphosphorylation, release of E2F/DP1, and increased expression of cyclin A.

20

Increasing cellular concentrations of cyclin D1/CDK4 complexes can also titrate CDK
inhibitors from the CDK2 complexes. Together, the increased levels of cyclins, and the
decreased association of CDK inhibitors lead to CDK2 activation (89, 90). Activated
CDK2 targets proteins involved in DNA replication (73). CDK2 also further
phosphorylates pRB, and causes the release of the E2F/DP family transcription factors,
and transcription of genes required for S phase entry. Various proteins involved in CDK
regulation, such as cyclin D1 and CDK inhibitors, have been identified as potential
mediators of E2-induced rnitogenesis, and deregulated expression of these key targets
may enable cells to proliferate in the absence of E2 or the presence of antiestrogens (108-

110).

The Protooncogene c-myc

The protooncogene c-myc is a key E2 target in breast cancer cells, and can
impinge upon the cell cycle machinery. The myc gene was identified over 25 years ago
as the oncogene v-myc of the MC29 avian melogytomatosis virus (111, 112). The c-myc
gene was originally isolated as the chicken cellular homologue of v-myc, which was
followed by the cloning and characterization of the human, mouse and rat c-myc genes
(113-116). Shortly thereafter, it was determined that oncogenic, activated c-myc is a key
agent in the etiology of human Burkitt's lymphoma (117). Aberrant c-myc expression
was also observed in a variety of human tumors including small cell lung carcinomas,
breast, cervical, and colon carcinomas, myeloid leukemias, melanomas, osteosarcomas,
and glioblastomas (118). Subsequently, two additional genes, N-myc and L-myc, were

identified as amplified myc-related genes in human geuroblastoma and small cell lung

21

carcinoma, respectively (119, 120). The myc family presently consists of three well-
characterized protooncogenes: c-myc, N-myc, L-myc, and two other members, B-myc and
S-myc, which have been identified only in rodents (121, 122). The c-myc, N-myc, L-myc
genes have similar genomic organization, and the corresponding proteins contain several
regions of high sequence homology.

The human c-myc gene, located on chromosome 8 (123), is comprised of three
exons: an untranslated exon (exon I) and two protein coding exons (exon H and 1H). The
c-myc gene has 2 major promoters, P1 and P2, located 161 nucleotides apart within the
exon I (Figure 5), and transcription initiated at these sites accounts for 5 - 10 and 85— 90
% of total c-myc mRNA respectively. Two other promoters: P0, located 500 bp upstream
of P1, and P3, located in intron 1, give rise to less then 5 % of c-myc mRNA each (124,
125). Both P1 and P2 contain a consensus TATA box element; however a strong
consensus initiator element (Inr) occurs only at the P2 transcription start site. A number
of different regulatory sequences have been described in the 5' upstream promoter region,
in the 5' and 3' untranslated regions, and within intron 1.

The c—myc gene is transcribed to give three major transcripts that start from
different initiation sites, yielding three proteins termed c-Mycl, c-Myc2, and c-MycS
(126). c-Myc2 is an approximately 62 kD protein that is the main isofonn and will
henceforth be referred to as ‘c-Myc’. There is only one open reading frame in all c-myc
mRNA species; it starts at the canonical AUG initiation codon located at the 5' end of
exon II and codes for c-Myc. c-Mycl arises from an alternative initiation site at an in-
frame CUG codon at the 3' end of exon 1, yielding a protein that is 2-4 kD larger than c-

Myc (127). c-MycS initiates at two closely spaced downstream AUG codons, resulting

22

 

 

 

 

B.
1 45 63 129 143 320 328 355 368 410 439
M31 MBlI NLS B HLH LZ
\ jk / \ J
V V Y
N - terminal Central region C - terminal
Transactivation Dimerization
DNA binding

Figure 5: Schematic representation of the c-myc gene organization and domain
structure of the c-Myc protein. A) (adapted from Ryan K.M. and Bernie G.D. Biochem
J. 1996; 314: 713-21. Figure 1). The c-myc gene is comprised of three exons. Exons 1,2,
and 3 are represented by open rectangles. The shaded region shows the untranslated
portion of the exon 1. Transcription start sites, P0, P1, P2, and P3 are indicated. The
translation initiation sites for c—Mycl — CTG (CUG) and c-Myc2 - ATG (AUG) are also
shown. B) (adapted from Pelengaris S. et al. Nat Rev Cancer. 2002; 2(10): 764-76.
Figure l). The c-Myc protein is composed of several functional domains. The amino
terminus harbors conserved ‘Myc homology boxes’ I and II (MBI and MBII), which are
essential for the transactivation of c-Myc target genes. The central region contains a
nuclear localization sequence (NLS). The carboxyl terminus contains the basic (B) helix-
loop-helix (HLH) leucine zipper (LZ) motif required for dimerization with its partner
Max and subsequent DNA binding of Myc-Max heterodimers.

23

Increasing cellular concentrations of cyclin D1/CDK4 complexes can also titrate CDK
inhibitors from the CDK2 complexes. Together, the increased levels of cyclins, and the
decreased association of CDK inhibitors lead to CDK2 activation (89, 90). Activated
CDK2 targets proteins involved in DNA replication (73). CDK2 also further
phosphorylates pRB, and causes the release of the E2F/DP family transcription factors,
and transcription of genes required for S phase entry. Various proteins involved in CDK
regulation, such as cyclin D1 and CDK inhibitors, have been identified as potential
mediators of E2-induced mitogenesis, and deregulated expression of these key targets
may enable cells to proliferate in the absence of E2 or the presence of antiestrogens (108-

110).

The Protooncogene c-myc

The protooncogene c-myc is a key E2 target in breast cancer cells, and can
impinge upon the cell cycle machinery. The myc gene was identified over 25 years ago
as the oncogene v-myc of the MC29 avian myelogytomatosis virus (111, 112). The c-myc
gene was originally isolated as the chicken cellular homologue of v-myc, which was
followed by the cloning and characterization of the human, mouse and rat c-myc genes
(113-116). Shortly thereafter, it was determined that oncogenic, activated c-myc is a key
agent in the etiology of human Burkitt's lymphoma (117). Aberrant c-myc expression
was also observed in a variety of human tumors including small cell lung carcinomas,
breast, cervical, and colon carcinomas, myeloid leukemias, melanomas, osteosarcomas,
and glioblastomas (118). Subsequently, two additional genes, N-myc and L-myc, were

identified as amplified myc-related genes in human geuroblastoma and small cell lung

21

carcinoma, respectively (119, 120). The myc family presently consists of three well-
characterized protooncogenes: c-myc, N-myc, L-myc, and two other members, B-myc and
S-myc, which have been identified only in rodents (121, 122). The c-myc, N-myc, L-myc
genes have similar genomic organization, and the corresponding proteins contain several
regions of high sequence homology.

The human c-myc gene, located on chromosome 8 (123), is comprised of three
exons: an untranslated exon (exon I) and two protein coding exons (exon II and H1). The
c—myc gene has 2 major promoters, P1 and P2, located 161 nucleotides apart within the
exon I (Figure 5), and transcription initiated at these sites accounts for 5 - 10 and 85— 9O
% of total c-myc mRN A respectively. Two other promoters: P0, located 500 bp upstream
of P1, and P3, located in intron I, give rise to less then 5 % of c-myc mRNA each (124,
125). Both P1 and P2 contain a consensus TATA box element; however a strong
consensus initiator element (Inr) occurs only at the P2 transcription start site. A number
of different regulatory sequences have been described in the 5' upstream promoter region,
in the 5' and 3' untranslated regions, and within intron 1.

The c-myc gene is transcribed to give three major transcripts that start from
different initiation sites, yielding three proteins termed c-Mycl, c-Myc2, and c-MycS
(126). c-Myc2 is an approximately 62 kD protein that is the main isoforrn and will
henceforth be referred to as ‘c-Myc’. There is only one open reading frame in all c-myc
mRNA species; it starts at the canonical AUG initiation codon located at the 5' end of
exon 11 and codes for c-Myc. c-Mycl arises from an alternative initiation site at an in-
frame CUG codon at the 3' end of exon 1, yielding a protein that is 2-4 kD larger than c-

Myc ( 127). c—MycS initiates at two closely spaced downstream AUG codons, resulting

22

 

 

 

 

B.
1 45 63 129 143 320 328 355 368 410 439
MBI MBIl NLS B HLH LZ
y j\ J\ J
V V Y
N - terminal Central region C - terminal
Transactivation Dimerization
DNA binding

Figure 5: Schematic representation of the c-myc gene organization and domain
structure of the c-Myc protein. A) (adapted from Ryan K.M. and Bernie G.D. Biochem
J. 1996; 314: 713-21. Figure 1). The c-myc gene is comprised of three exons. Exons 1,2,
and 3 are represented by open rectangles. The shaded region shows the untranslated
portion of the exon 1. Transcription start sites, P0, P1, P2, and P3 are indicated. The
translation initiation sites for c-Mycl - CTG (CUG) and c-Myc2 - ATG (AUG) are also
shown. B) (adapted from Pelengaris S. et al. Nat Rev Cancer. 2002; 2(10): 764-76.
Figure 1). The c-Myc protein is composed of several functional domains. The amino
terminus harbors conserved ‘Myc homology boxes’ I and II (MBI and MBII), which are
essential for the transactivation of c-Myc target genes. The central region contains a
nuclear localization sequence (N LS). The carboxyl terminus contains the basic (B) helix-
loop-helix (HLH) leucine zipper (LZ) motif required for dimerization with its partner
Max and subsequent DNA binding of Myc-Max heterodimers.

23

in a protein missing about 100 amino acids at the N-terminus of c-Myc, and lacks the
transactivation ability of c-Myc (128).

The c-Myc protein has several structural domains, which are depicted in Figure 5.
The first 143 amino acids of the amino terminus comprise the transactivation domain
(TAD) that contains two regions called Myc homology boxes (MBI and MBII), which are
highly conserved among members of the Myc family. MBI is required for the
transactivation activities of c-Myc, whereas MBII is needed for transrepression. c-Myc
also has a nuclear localization sequence (NLS) at amino acids 320-328 (129). The
carboxyl terminus of c-Myc contains the basic region (B) (amino acids 355-368)
implicated in specific DNA sequence recognition and binding. It also includes the helix-
loop-helix/leucine zipper (HLH/Ll) region (amino acids 368-439), which is responsible
for the specific heterodimer formation between c-Myc and its binding partner, another
transcription factor called Max (c-m_yc _associated protein 5) (130). Contiguous BR-HLH-
LZ motifs are characteristic of transcription factors that bind to specific DNA sequences.
The c-Myc/Max complexes bind to a specific DNA recognition sequence, the E box
element, that contains a central CAC(G/A)TG motif. Genes containing the E box
element in their regulatory regions may be subjected to transactivation or transrepression
by the c-Myc/Max complexes, whereas Max-Max or Max-Mad dimers, may block the
biological effects of Myc-Max dimers by competitive occupancy of these binding sites

(131-133).

24

c-Myc and Cell Cycle Regulation

One of the key biological firnctions of c-Myc is to promote cell cycle progression.
Other than mediating proliferation, c-Myc is also known to play an important role in
differentiation and apoptosis. Suppression of c-Myc may be necessary for differentiation
of certain cell types, perhaps because withdrawal from the cell cycle via c-Myc down-
regulation is essential for terminal differentiation. However, some studies show that the
ability of c-Myc to block cell differentiation can be uncoupled from its ability to drive
proliferation (134, 135). With regard to apoptosis, there are two views regarding why c-
Myc, which is a potent inducer of cell proliferation, also possesses apoptotic activity.
One interpretation is that oncogenes such as c-Myc activate apoptosis if the proliferative
pathway is blocked in some way. The other, more widely held, view is that induction of
cell cycle entry sensitizes the cell to apoptosis, but the apoptotic pathway is suppressed as
long as appropriate survival factors deliver anti-apoptotic signals. The outcome of these
contradictory processes thus depends on the availability of survival factors (130). Given
its physiological roles, it is not surprising that c-Myc also has a role in carcinogenesis and
cellular transformation (126).

c-Myc was first implicated in human cancers over two decades ago, and since
then significant effort has been put into identifying targets of c-Myc. Recent
developments in high-throughput assays such as microarrays, SAGE, and scanning
chromatin immunoprecipitation have led to the discovery of a vast collection of c-Myc-
responsive genes, and have resulted in the establishment of an integrated database called
the “Myc Target Gene Database”. An international advisory board provides oversight of

the contents of this database, which are updated quarterly. Based on various putative

25

target genes identified, c-Myc is now implicated in cellular functions other than
proliferation, differentiation and apoptosis. These processes include ribosome
biogenesis, protein synthesis and various metabolic pathways, and a potential
involvement of c-Myc in these processes is in agreement with its established role in
stimulating cell grth (118, 136).

Despite the identification of many targets, a key role for c-Myc remains the
promotion of cell proliferation. The studies that we have carried out focus on breast
cancer cells. In other systems, among the numerous targets of c-Myc that have been
identified are various cell cycle regulatory proteins, which are likely to be involved in
mediating c-Myc’s proliferative effects (137, 138). c-Myc could be mediating its
proliferative effects by increasing the levels of positive cell cycle regulators, decreasing
the levels of cell cycle inhibitors, and affecting CDK complex formation and/or kinase
activities. c-Myc increases the mRNA levels of various positive cell cycle regulators
including cyclin D2, CDK4 and Cdc25. c-Myc also transcriptionally uprcgulates Id2, a
helix-loop-lielix protein that binds to hypophosphorylated pRB and causes the loss of
pRB-mediated growth suppression, and this in turn may facilitate cyclin B transcription
(139). An effect of c-Myc on cyclin D1 transcription is somewhat controversial, since c-
Myc has been shown to either increase, decrease or have no effect on cyclin D1
expression (110, 140-142). c-Myc could also mediate proliferation by decreasing the
levels of CDK inhibitors and affecting their distribution among G1 cyclin complexes. It
has been shown to repress p21 transcriptionally and mediate degradation of the p27
protein (143-148). In addition, there is evidence that activation of cyclin E-Cdk2 is an

essential step in c-Myc-induced Gl-S progression (149), and expression of c-Myc in

26

breast cancer cells using an inducible system, has shown that CDK2 rather than CDK4

activation is seen upon c-Myc expression (110).

Estrogen and c-Myc

c-myc mRNA is rapidly induced in E2-responsive breast cancer cells following
E2 treatment. Pretreatment with the protein synthesis inhibitor cyclohexirnide does not
prevent c-Myc induction, indicating that c-Myc is a direct target of E2 (150-153).
Transient transfections with c-myc promoter-reporter gene constructs have shown that
116 bp, including the TATA box and upstream regions of the P2 promoter, are necessary
for E2-regulated reporter gene activity. This E2-reponsive region lacks any consensus
perfect or imperfect ERE. Though this suggests that ER acts at the c—myc promoter in an
ERE-independent manner such as through protein-protein interactions with AP-l
transcription factor; co-transfection studies with mutant ER expression vectors have
shown that the DNA binding domain of the receptor is essential for E2-regulated reporter
gene activity, suggesting that the ER complex may bind sequences in the 116 bp region
(154). Recent studies using Chromatin immunoprecipitation (ChIP) assays have
corroborated the above studies by demonstrating that E2 treatment of breast cancer cells
leads to ER binding and co-activator recruitment at the c-myc promoter (155). Thus, a
model has emerged in which E2-liganded ER binds to the proximal sequences in the c-
myc promoter, recruits co-activators, and leads to rapid induction of c-Myc. This
induction of c-Myc is required for E2’s effects on cell cycle progression, as c-myc
antisense oligonucleotides can suppress E2-stimulated breast cancer cell proliferation. In

addition, ectopic expression of c-Myc is sufficient to induce cell cycle progression of E2-

27

dependent breast cancer cells in the presence of antiestrogens (110, 156-158). Thus, c-
myc plays an essential role in mediating E2-regulated proliferation, and can contribute to

antiestrogen-resistant proliferation of breast cancer cells.

Mechanisms of Antiestrogen Resistance

Various mechanisms have been proposed to explain the progression of tumors to
an antiestrogen-resistant phenotype. These include loss or mutation of the ER, altered
antiestrogen metabolism, a switch to an antiestrogen-stimulated phenotype, aberrant
expression of ER coactivators/corepressors, and deregulation of protooncogenes like v-H-
ras, c-myc and c-jun (110, 156, 159-161). These are not proposed to be mutually
exclusive mechanisms.

Although loss of ER could result in antiestrogen resistance prior to treatment,
resistance acquired during the course of treatment is not commonly associated with loss
of ER expression or with ER mutations (162). Altered antiestrogen metabolism has not
been shown to be a likely cause of antiestrogen resistance, and using indirect evidence it
has been estimated that a switch to a Tamoxifen-stimulated phenotype occurs in less than
20% of initially sensitive tumors (159, 163-165). As discussed previously, the
coactivator AIBl has been implicated in Tamoxifen resistance, and the role of altered
coactivator or corepressor expression in antiestrogen resistance is under intense
investigation. However these analyses are confounded due to the functional redundancy
of these coregulators.

Ultimately, since E2 and antiestrogens control cell cycle progression, the

development of resistance must be associated with changes in cell cycle regulation.

28

Numerous laboratories have therefore focused on the possibility that resistance can result
from the deregulated expression and/or activity of either cell cycle proteins or gene
products that can target cell cycle regulators. It has been shown that induction of positive
cell cycle regulators such as cyclin D1 and cyclin B can lead to antiestrogen-resistant
proliferation of breast cancer cells. Inhibiting expression of negative regulators such as
p21 and p27, using antisense oligonucleotides, or inactivation of pRB with T-antigen, is
also sufficient to overcome an ICI arrest (108-110, 166-169). We focused on studying
the proto-oncogene product c-Myc, since it is a direct target of E2 that is important for
E2-stimulated proliferation, and because c-Myc expression alone is sufficient to induce
cell cycle progression in the presence of antiestrogens (110, 150, 151, 154, 156-158).
c-Myc has been extensively studied in many systems, however the target(s)
through which it mediates E2-stimulated proliferation, and contributes to cell cycle
progression in the presence of antiestrogens in breast cancer cells, have not been
identified. The goal of this dissertation work was to identify these target(s), and to
determine whether aberrant regulation of c-Myc and/or its target(s) is associated with
acquired antiestrogen resistance, using in vitro models of human breast cancer. The

studies carried out to address these goals are described in the subsequent chapters.

29

10.

11.

REFERENCES

Parkin, D. M., Bray, F., Ferlay, J ., and Pisani, P. Global cancer statistics, 2002.
CA Cancer J Clin, 55: 74-108, 2005.

Jemal, A., Clegg, L. X., Ward, E., Ries, L. A., Wu, X., Jamison, P. M., Wingo, P.
A., Howe, H. L., Anderson, R. N., and Edwards, B. K. Annual report to the nation
on the status of cancer, 1975-2001, with a special feature regarding survival.
Cancer, 101: 3-27, 2004.

Feuer, E. J., Wun, L. M., Boring, C. C., Flanders, W. D., Tirnmel, M. J ., and
Tong, T. The lifetime risk of developing breast cancer. J Natl Cancer Inst, 85 :
892-897, 1993.

Gail, M. H., Brinton, L. A., Byar, D. P., Corle, D. K., Green, S. B., Schairer, C.,
and Mulvihill, J. J. Projecting individualized probabilities of developing breast
cancer for white females who are being exanrined annually. J Natl Cancer Inst,
81: 1879-1886, 1989.

Pike, M. C., Krailo, M. D., Henderson, B. E., Casagrande, J. T., and Hoel, D. G.
'Horrnonal' risk factors, 'breast tissue age‘ and the age-incidence of breast cancer.
Nature, 303: 767-770, 1983.

Russo, J. and Russo, I. H. The etiopathogenesis of breast cancer prevention.
Cancer Lett, 90: 81-89, 1995.

Landis, S. H., Murray, T., Bolden, S., and Wingo, P. A. Cancer statistics, 1999.
CA Cancer J Clin, 49: 8-31, 31, 1999.

Haslam, S. Z., Osuch, J. R., Raafat, A. M., and Hofseth, L. J. Postmenopausal
hormone replacement therapy: effects on normal mammary gland in humans and

in a mouse postmenopausal model. J Mammary Gland Biol Neoplasia, 7: 93-105,
2002.

Reed, M. J. The role of aromatase in breast tumors. Breast Cancer Res Treat, 30:
7-17, 1994.

Laidlaw, I. J ., Clarke, R. B., Howell, A., Owen, A. W., Potten, C. S., and
Anderson, E. The proliferation of normal human breast tissue implanted into

athymic nude mice is stimulated by estrogen but not progesterone. Endocrinology,
136: 164-171, 1995.

Russo, I. H. and Russo, J. Role of hormones in mammary cancer initiation and
progression. J Mammary Gland Biol Neoplasia, 3: 49-61, 1998.

30

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

Jefcoate, C. R., Liehr, J. G., Santen, R. J ., Sutter, T. R., Yager, J. D., Yue, W.,
Santrrer, S. J ., Tekrnal, R., Demers, L., Pauley, R., Naftolin, F., Mor, G., and

Berstein, L. Tissue-specific synthesis and oxidative metabolism of estrogens. J
Natl Cancer Inst Monogr 95-112, 2000.

Yager, J. D. Endogenous estrogens as carcinogens through metabolic activation. J
Natl Cancer Inst Monogr 67-73, 2000.

Dickey, R. P. and Stone, S. C. Drugs that affect the breast and lactation. Clin
Obstet Gynecol, 18: 95-111, 1975.

Iversen, S. A target-theory interpretation of the dose-response relationship
between oestrogen and functional differentiation of the mammary gland in spayed
mice. J Endocrinol, 17: 99-106, 1958.

Hirohashi, S., Shimosato, Y., Kameya, T., Nagai, K., and Tsunematsu, R.
Hormone dependency of a serially transplantable human breast cancer (Br-10) in
nude mice. Cancer Res, 3 7: 3184-3189, 1977.

Shafie, S. M. and Grantham, F. H. Role of hormones in the growth and regression
of human breast cancer cells (MCF-7) transplanted into athymic nude mice. J Natl
Cancer Inst, 6 7: 51-56, 1981.

Soule, H. D. and McGrath, C. M. Estrogen responsive proliferation of clonal
human breast carcinoma cells in athymic mice. Cancer Lett, 10: 177-189, 1980.

Rutqvist, L. E., Cedermark, B., Glas, U., J ohansson, H., Nordenskjold, B., Skoog,
L., Somell, A., Theve, T., Friberg, S., and Askergren, J. The Stockhohn trial on
adjuvant tamoxifen in early breast cancer. Correlation between estrogen receptor
level and treatment effect. Breast Cancer Res Treat, 10: 255-266, 1987.

Partridge, A. H., Burstein, H. J ., and Winer, E. P. Side effects of chemotherapy
and combined chemohormonal therapy in women with early-stage breast cancer. J
Natl Cancer Inst Monogr 135-142, 2001.

Systemic treatment of early breast cancer by hormonal, cytotoxic, or immune
therapy. 133 randomised trials involving 31,000 recurrences and 24,000 deaths
among 75,000 women. Early Breast Cancer Trialists' Collaborative Group.
Lancet, 339: 1-15, 1992.

Tamoxifen for early breast cancer: an overview of the randomised trials. Early
Breast Cancer Trialists' Collaborative Group. Lancet, 351 : 1451-1467, 1998.

Fisher, B., Costantino, J. P., Wickerham, D. L., Redmond, C. K., Kavanah, M.,

Cronin, W. M., Vogel, V., Robidoux, A., Dirnitrov, N., Atkins, J ., Daly, M.,
Wieand, S., Tan-Chiu, E., Ford, L., and Wolrnark, N. Tamoxifen for prevention of

31

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

breast cancer: report of the National Surgical Adjuvant Breast and Bowel Project
P-l Study. J Natl Cancer Inst, 90: 1371-1388, 1998.

Prowell, T. M. and Davidson, N. E. What is the role of ovarian ablation in the

management of primary and metastatic breast cancer today? Oncologist, 9: 507-
5 l 7, 2004.

Kistner, R. W. and Smith, 0. W. Observations on the use of a non-steroidal
estrogen antagonist: MER-25. Surg Forum, 10: 725-729, 1960.

Cole, M. P., Jones, C. T., and Todd, 1. D. A new anti-oestrogenic agent in late
breast cancer. An early clinical appraisal of ICI46474. Br J Cancer, 25 : 270-275,
1971.

Wakeling, A. E. Similarities and distinctions in the mode of action of different
classes of antioestrogens. Endocr Relat Cancer, 7: 17-28, 2000.

Lonard, D. M. and Smith, C. L. Molecular perspectives on selective estrogen
receptor modulators (SERMs): progress in understanding their tissue-specific
agonist and antagonist actions. Steroids, 6 7: 15-24, 2002.

DeFriend, D. J ., Anderson, E., Bell, J ., Wilks, D. P., West, C. M., Manse], R. E.,
and Howell, A. Effects of 4-hydroxytamoxifen and a novel pure antioestrogen

(ICI 182780) on the clonogenic growth of human breast cancer cells in vitro. Br J
Cancer, 70: 204-211, 1994.

DeFriend, D. J ., Howell, A., Nicholson, R. 1., Anderson, R, Dowsett, M., Mansel,
R. E., Blamey, R. W., Bundred, N. J ., Robertson, J. F., Saunders, C., and et al.
Investigation of a new pure antiestrogen (ICI 182780) in women with primary
breast cancer. Cancer Res, 54: 408-414, 1994.

Dauvois, S., Danielian, P. S., White, R., and Parker, M. G. Antiestrogen ICI
164,384 reduces cellular estrogen receptor content by increasing its turnover. Proc
Natl Acad Sci U S A, 89: 4037-4041, 1992.

Johnston, S. R. and Dowsett, M. Aromatase inhibitors for breast cancer: lessons
from the laboratory. Nat Rev Cancer, 3: 821-831, 2003.

Baum, M., Budzar, A. U., Cuzick, J ., Forbes, J ., Houghton, J. H., Klijn, J. G., and
Sahmoud, T. Anastrozole alone or in combination with tamoxifen versus
tamoxifen alone for adj uvant treatment of postmenopausal women with early
breast cancer: first results of the ATAC randomised trial. Lancet, 359: 2131-2139,
2002.

Howell, A., Cuzick, J ., Baum, M., Buzdar, A., Dowsett, M., Forbes, J. F ., Hoctin-
Boes, G., Houghton, J ., Locker, G. Y., and Tobias, J. S. Results of the ATAC

32

35.

36.

37.

38.

39.

40.

41.

42.

43.

(Arimidex, Tamoxifen, Alone or in Combination) trial afler completion of 5 years'
adjuvant treatment for breast cancer. Lancet, 365 : 60-62, 2005.

Wolf, D. M. and Jordan, V. C. William L. McGuire Memorial Symposium. Drug
resistance to tamoxifen during breast cancer therapy. Breast Cancer Res Treat, 27:
27-40, 1993.

Long, B. J ., Jelovac, D., Thiantanawat, A., and Brodie, A. M. The effect of
second-line antiestro gen therapy on breast tumor growth after first-line treatment
with the aromatase inhibitor letrozole: long-term studies using the intratumoral

aromatase postmenopausal breast cancer model. Clin Cancer Res, 8: 23 78-2388,
2002.

Vergote, I., Robertson, J. F ., Kleeberg, U., Burton, G., Osborne, C. K., and
Mauriac, L. Postmenopausal women who progress on fulvestrant ('Faslodex')

remain sensitive to further endocrine therapy. Breast Cancer Res Treat, 79: 207-
21 1, 2003.

Chlebowski, R. T., Col, N., Winer, E. P., Collyar, D. E., Cummings, S. R., Vogel,
V. G., 3rd, Burstein, H. J ., Eisen, A., Lipkus, I., and Pfister, D. G. American
Society of Clinical Oncology technology assessment of pharmacologic

interventions for breast cancer risk reduction including tamoxifen, raloxifene, and
aromatase inhibition. J Clin Oncol, 20: 3328-3343, 2002.

Chlebowski, R. T., Collyar, D. E., Somerfield, M. R., and Pfister, D. G. American
Society of Clinical Oncology technology assessment on breast cancer risk
reduction strategies: tamoxifen and raloxifene. J Clin Oncol, I 7: 1939-1955,
1999.

Mosselrnan, S., Polrnan, J ., and Dijkema, R. ER beta: identification and

characterization of a novel human estrogen receptor. FEBS Lett, 392: 49-53,
1996.

Couse, J. F., Lindzey, J ., Grandien, K., Gustafsson, J. A., and Korach, K. S.
Tissue distribution and quantitative analysis of estrogen receptor-alpha (ERalpha)
and estrogen receptor-beta (ERbeta) messenger ribonucleic acid in the wild-type
and ERalpha-knockout mouse. Endocrinology, 138: 4613-4621, 1997.

Couse, J. F. and Korach, K. S. Estrogen receptor null mice: what have we learned
and where will they lead us? Endocr Rev, 20: 358-417, 1999.

Tora, L., White, J ., Brou, C., Tasset, D., Webster, N., Scheer, E., and Charnbon,

P. The human estrogen receptor has two independent nonacidic transcriptional
activation functions. Cell, 59: 477-487, 1989.

33

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

Kumar, V., Green, S., Stack, G., Berry, M., J in, J. R., and Chambon, P. Functional
domains of the human estrogen receptor. Cell, 51 : 941-951, 1987.

Hall, J. M., Couse, J. F., and Korach, K. S. The multifaceted mechanisms of
estradiol and estrogen receptor signaling. J Biol Chem, 276: 36869-36872, 2001.

Klein-Hitpass, L., Ryffel, G. U., Heitlinger, E., and Cato, A. C. A 13 bp
palindrome is a functional estrogen responsive element and interacts specifically
with estrogen receptor. Nucleic Acids Res, 16: 647-663, 1988.

Klein-Hitpass, L., Tsai, S. Y., Greene, G. L., Clark, J. H., Tsai, M. J ., and
O'Malley, B. W. Specific binding of estrogen receptor to the estrogen response
element. Mol Cell Biol, 9: 43-49, 1989.

Kumar, V. and Chambon, P. The estrogen receptor binds tightly to its responsive
element as a ligand-induced homodimer. Cell, 55: 145-156, 1988.

Gaub, M. P., Bellard, M., Scheuer, I., Chambon, P., and Sassone-Corsi, P.
Activation of the ovalan gene by the estrogen receptor involves the fos-jun
complex. Cell, 63: 1267-1276, 1990.

Safe, 8. Transcriptional activation of genes by 17 beta-estradiol through estrogen
receptor-Spl interactions. Vitarn Horm, 62: 231-252, 2001.

El-Tanarri, M. K. and Green, C. D. Two separate mechanisms for ligand-
independent activation of the estrogen receptor. Mol Endocrinol, 11: 928-937,
1 997 .

Sirnoncini, T., Hafezi-Moghadam, A., Brazil, D. P., Ley, K., Chin, W. W., and
Liao, J. K. Interaction of oestrogen receptor with the regulatory subunit of
phosphatidylinositol-3-OH kinase. Nature, 407: 538-541, 2000.

Kousteni, S., Bellido, T., Plotkin, L. I., O'Brien, C. A., Bodenner, D. L., Han, L.,
Han, K., DiGregorio, G. B., Katzenellenbogen, J. A., Katzenellenbogen, B. S.,
Roberson, P. K., Weinstein, R. S., Jilka, R. L., and Manolagas, S. C.
Nongenotropic, sex-nonspecific signaling through the estrogen or androgen
receptors: dissociation from transcriptional activity. Cell, 104: 719-730, 2001.

Benten, W. P., Stephan, C., Lieberherr, M., and Wunderlich, F. Estradiol
signaling via sequestrable surface receptors. Endocrinology, 142: 1669-1677,
2001.

Bjomstrom, L. and Sjoberg, M. Mechanisms of estrogen receptor signaling:
convergence of genomic and nongenomic actions on target genes. Mol
Endocrinol, 19: 833-842, 2005.

34

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

Gruber, C. J ., Tschugguel, W., Schneeberger, C., and Huber, J. C. Production and
actions of estrogens. N Engl J Med, 346: 340-352, 2002.

Graham, J. D., Bain, D. L., Richer, J. K., Jackson, T. A., Tung, L., and Horwitz,
K. B. Nuclear receptor conformation, coregulators, and tamoxifen-resistant breast
cancer. Steroids, 65: 579-584, 2000.

Dutertre, M. and Smith, C. L. Molecular mechanisms of selective estrogen
receptor modulator (SERM) action. J Pharmacol Exp Ther, 295: 431—437, 2000.

Edwards, D. P. The role of coactivators and corepressors in the biology and
mechanism of action of steroid hormone receptors. J Mammary Gland Biol
Neoplasia, 5: 307-324, 2000.

Smith, C. L. and O'Malley, B. W. Coregulator function: a key to understanding
tissue specificity of selective receptor modulators. Endocr Rev, 25: 45-71, 2004.

Xu, J. and Li, Q. Review of the in vivo functions of the p160 steroid receptor
coactivator family. Mol Endocrinol, I 7: 1681-1692, 2003.

Xu, J ., Qiu, Y., DeMayo, F. J ., Tsai, S. Y., Tsai, M. J ., and O'Malley, B. W.
Partial hormone resistance in mice with disruption of the steroid receptor
coactivator-1 (SRC-1) gene. Science, 279: 1922-1925, 1998.

Anzick, S. L., Kononen, J ., Walker, R. L., Azorsa, D. 0., Tanner, M. M., Guan,
X. Y., Sauter, G., Kallioniemi, O. P., Trent, J. M., and Meltzer, P. S. AlBl, a

steroid receptor coactivator amplified in breast and ovarian cancer. Science, 277:
965-968, 1997.

Louie, M. C., Zou, J. X., Rabinovich, A., and Chen, H. W. ACTR/AIBl functions
as an E2F1 coactivator to promote breast cancer cell proliferation and
antiestrogen resistance. Mol Cell Biol, 24: 5157-5171, 2004.

Osborne, C. K., Bardou, V., Hopp, T. A., Charnness, G. C., Hilsenbeck, S. G.,
Fuqua, S. A., Wong, J ., Allred, D. C., Clark, G. M., and Schiff, R. Role of the
estrogen receptor coactivator AIBl (SRC-3) and HER-2/neu in tamoxifen
resistance in breast cancer. J Natl Cancer Inst, 95: 353-361, 2003.

Heichman, K. A. and Roberts, J. M. Rules to replicate by. Cell, 79: 557-562,
1994.

Laskey, R. A., Fairman, M. P., and Blow, J. J. S phase of the cell cycle. Science,
246: 609-614, 1989.

McIntosh, J. R. and Koonce, M. P. Mitosis. Science, 246: 622-628, 1989.

35

69.

70.

71.

72.

73.

74.

75.

76.

77.

78.

79.

80.

81.

82.

83.

Stilhnan, B. Cell cycle control of DNA replication. Science, 274: 1659-1664,
1996.

Hartwell, L. H. and Weinert, T. A. Checkpoints: controls that ensure the order of
cell cycle events. Science, 246: 629-634, 1989.

Pardee, A. B. G1 events and regulation of cell proliferation. Science, 246: 603-
608, 1989.

Pardee, A. B. A restriction point for control of normal animal cell proliferation.
Proc Natl Acad Sci U S A, 71: 1286-1290, 1974.

Sherr, C. J. Cancer cell cycles. Science, 274: 1672-1677, 1996.
Morgan, D. 0. Principles of CDK regulation. Nature, 374: 131-134, 1995.
Hunter, T. and Pines, J. Cyclins and cancer. Cell, 66: 1071-1074, 1991.

Hunter, T. and Pines, J. Cyclins and cancer. 11: Cyclin D and CDK inhibitors
come of age. Cell, 79: 573-582, 1994.

Sherr, C. J. D-type cyclins. Trends Biochem Sci, 20: 187-190, 1995.

van den Heuvel, S. and Harlow, E. Distinct roles for cyclin-dependent kinases in
cell cycle control. Science, 262: 2050-2054, 1993.

Sherr, C. J. and Roberts, J. M. Inhibitors of mammalian G1 cyclin-dependent
kinases. Genes Dev, 9: 1149-1163, 1995.

Serrano, M., Hannon, G. J ., and Beach, D. A new regulatory motif in cell-cycle
control causing specific inhibition of cyclin D/CDK4. Nature, 366: 704-707,
1993.

Xiong, Y., Hannon, G. J ., Zhang, H., Casso, D., Kobayashi, R., and Beach, D. p21
is a universal inhibitor of cyclin kinases. Nature, 366: 701-704, 1993.

Polyak, K., Lee, M. H., Erdjument-Bromage, H., Koff, A., Roberts, J. M.,
Tempst, P., and Massague, J. Cloning of p27Kipl , a cyclin-dependent kinase
inhibitor and a potential mediator of extracellular antimitogenic signals. Cell, 78:
59-66, 1994.

Toyoshima, H. and Hunter, T. p27, a novel inhibitor of G1 cyclin-Cdk protein
kinase activity, is related to p21. Cell, 78: 67-74, 1994.

36

84.

85.

86.

87.

88.

89.

90.

91.

92.

93.

94.

LaBaer, J ., Garrett, M. D., Stevenson, L. F., Slingerland, J. M., Sandhu, C., Chou,
H. S., Fattaey, A., and Harlow, E. New functional activities for the p21 family of
CDK inhibitors. Genes Dev, 11: 847-862, 1997.

Cheng, M., Olivier, P., Diehl, J. A., Fero, M., Roussel, M. F., Roberts, J. M., and
Sherr, C. J. The p21(Cip1) and p27(Kipl) CDK 'inhibitors' are essential activators
of cyclin D-dependent kinases in murine fibroblasts. Embo J, 18: 1571-1583,
1999.

Weiss, R. H., J 00, A., and Random, C. p21Waf1/Cip1 is an assembly factor
required for platelet-derived growth factor-induced vascular smooth muscle cell
proliferation. J Biol Chem, 275: 28340, 2000.

Jaumot, M., Estanol, J. M., Casanovas, 0., Grana, X., Agell, N., and Bachs, O.
The cell cycle inhibitor p21CIP is phosphorylated by cyclin A-CDK2 complexes.
Biochem Biophys Res Commun, 241: 434-438, 1997.

Chiarle, R., Budel, L. M., Skolnik, J ., Frizzera, G., Chilosi, M., Corato, A.,
Pizzolo, G., Magidson, J ., Montagrroli, A., Pagano, M., Macs, B., De Wolf-
Peeters, C., and Inghirarni, G. Increased proteasome degradation of cyclin-
dependent kinase inhibitor p27 is associated with a decreased overall survival in
mantle cell lymphoma. Blood, 95: 619-626, 2000.

Planas-Silva, M. D. and Weinberg, R. A. Estrogen-dependent cyclin E-cdk2
activation through p21 redistribution. Mol Cell Biol, 1 7: 4059-4069, 1997 .

Prall, O. W., Sarcevic, B., Musgrove, E. A., Watts, C. K., and Sutherland, R. L.
Estrogen-induced activation of Cdk4 and Cdk2 during Gl-S phase progression is
accompanied by increased cyclin D1 expression and decreased cyclin-dependent
kinase inhibitor association with cyclin E-Cdk2. J Biol Chem, 272: 10882-10894,
1997 .

Gautier, J ., Solomon, M. J ., Booher, R. N., Bazan, J. F., and Kirschner, M. W.
cd025 is a specific tyrosine phosphatase that directly activates p34cdc2. Cell, 67:
197-211, 1991.

Dunphy, W. G. and Kumagai, A. The cdc25 protein contains an intrinsic
phosphatase activity. Cell, 67: 189-196, 1991.

Hoffrnann, 1., Draetta, G., and Karsenti, E. Activation of the phosphatase activity
of human cdc25A by a cdk2-cyclin E dependent phosphorylation at the G1/S
transition. Embo J, 13: 4302-4310, 1994.

Aprelikova, 0., Xiong, Y., and Lin, B. T. Both p16 and p21 families of cyclin-

dependent kinase (CDK) inhibitors block the phosphorylation of cyclin-dependent
kinases by the CDK-activating kinase. J Biol Chem, 270: 18195-18197, 1995.

37

95.

96.

97.

98.

99.

100.

101.

102.

103.

104.

105.

106.

Saha, P., Eichbaum, Q., Silberman, E. D., Mayer, B. J ., and Dutta, A. p21CIPl
and Cdc25A: competition between an inhibitor and an activator of cyclin-
dependent kinases. Mol Cell Biol, 1 7: 4338-4345, 1997.

Weinberg, R. A. The retinoblastoma protein and cell cycle control. Cell, 81: 323-
330, 1995.

Chellappan, S. P., Hiebert, S., Mudryj, M., Horowitz, J. M., and Nevins, J. R. The
E2F transcription factor is a cellular target for the RB protein. Cell, 65: 105 3-
1061, 1991.

DeGregori, J., Kowalik, T., and Nevins, J. R. Cellular targets for activation by the

E2F1 transcription factor include DNA synthesis- and GUS-regulatory genes.
Mol Cell Biol, 15: 4215-4224, 1995.

Harbour, J. W. and Dean, D. C. The Rb/E2F pathway: expanding roles and
emerging paradigms. Genes Dev, 14: 2393-2409, 2000.

Dyson, N. The regulation of E2F by pRB-family proteins. Genes Dev, 12: 2245-
2262, 1998.

Chittenden, T., Livingston, D. M., and DeCaprio, J. A. Cell cycle analysis of E2F
in primary human T cells reveals novel E2F complexes and biochemically distinct
forms of free E2F. Mol Cell Biol, 13: 3975-3983, 1993.

Leung, B. S. and Potter, A. H. Mode of estrogen action on cell proliferation in
CAMA-l cells: H. Sensitivity of G1 phase population. J Cell Biochem, 34: 213-
225, 1987.

Sutherland, R. L., Green, M. D., Hall, R. E., Reddel, R. R., and Taylor, I. W.
Tamoxifen induces accumulation of MCF 7 human mammary carcinoma cells in
the GO/Gl phase of the cell cycle. Eur J Cancer Clin Oncol, 19: 615-621, 1983.

Sutherland, R. L., Reddel, R. R., and Green, M. D. Effects of oestrogens on cell
proliferation and cell cycle kinetics. A hypothesis on the cell cycle effects of
antioestrogens. Eur J Cancer Clin Oncol, 19: 307-318, 1983.

Altucci, L., Addeo, R., Cicatiello, L., Dauvois, S., Parker, M. G., Truss, M.,
Beato, M., Sica, V., Bresciani, F., and Weisz, A. 17beta-Estradiol induces cyclin
D1 gene transcription, p36D1-p34cdk4 complex activation and p105Rb
phosphorylation during rrritogenic stimulation of G(1)-arrested human breast
cancer cells. Oncogene, 12: 2315-2324, 1996.

Foster, J. S. and Wimalasena, J. Estrogen regulates activity of cyclin-dependent
kinases and retinoblastoma protein phosphorylation in breast cancer cells. Mol
Endocrinol, 1 0: 488-498, 1996.

38

107.

108.

109.

110.

111.

112.

113.

114.

115.

116.

117.

Foster, J. S., Henley, D. C., Bukovsky, A., Seth, P., and Wimalasena, J.
Multifaceted regulation of cell cycle progression by estrogen: regulation of Cdk
inhibitors and Cdc25A independent of cyclin D1-Cdk4 function. Mol Cell Biol,
21 : 794-810, 2001.

Bindels, E. M., Lallemand, F., Balkenende, A., Verwoerd, D., and Michalides, R.
Involvement of G1/S cyclins in estrogen-independent proliferation of estrogen
receptor-positive breast cancer cells. Oncogene, 21: 8158-8165, 2002.

Cariou, 8., Donovan, J. C., Flanagan, W. M., Milic, A., Bhattacharya, N., and
Slingerland, J. M. Down-regulation of p21WAFl/CIP1 or p27Kip1 abrogates
antiestrogen-mediated cell cycle arrest in human breast cancer cells. Proc Natl
Acad Sci U S A, 97: 9042-9046, 2000.

Prall, O. W., Rogan, E. M., Musgrove, E. A., Watts, C. K., and Sutherland, R. L.
c-Myc or cyclin D1 mimics estrogen effects on cyclin E-Cdk2 activation and cell
cycle reentry. Mol Cell Biol, 18: 4499-4508, 1998.

Sheiness, D. and Bishop, J. M. DNA and RNA fi'om uninfected vertebrate cells
contain nucleotide sequences related to the putative transforming gene of avian
myelocytomatosis virus. J Virol, 31 : 514-521, 1979.

Sheiness, D., Fanshier, L., and Bishop, J. M. Identification of nucleotide

sequences which may encode the oncogenic capacity of avian retrovirus MC29. J
Virol, 28: 600-610, 1978.

Vennstrom, B., Sheiness, D., Zabielski, J ., and Bishop, J. M. Isolation and
characterization of c-myc, a cellular homolog of the oncogene (v-myc) of avian
myelocytomatosis virus strain 29. J Virol, 42: 773-779, 1982.

Dalla-Favera, R., Gelrnann, E. P., Martinotti, S., Franchini, G., Papas, T. S.,
Gallo, R. C., and Wong-Staal, F. Cloning and characterization of different human

sequences related to the onc gene (v-myc) of avian myelocytomatosis virus
(MC29). Proc Natl Acad Sci U S A, 79: 6497-6501, 1982.

Hayashi, K., Makino, R., Kawamura, H., Arisawa, A., and Yoneda, K.
Characterization of rat c-myc and adjacent regions. Nucleic Acids Res, 15: 6419-
6436, 1987 .

Stanton, L. W., Fahrlander, P. D., Tesser, P. M., and Marcu, K. B. Nucleotide
sequence comparison of normal and translocated murine c-myc genes. Nature,
310: 423-425, 1984.

Dalla-Favera, R., Bregni, M., Erikson, J ., Patterson, D., Gallo, R. C., and Croce,
C. M. Human c-myc one gene is located on the region of chromosome 8 that is

39

118.

119.

120.

121.

122.

123.

124.

125.

126.

127.

translocated in Burkitt lymphoma cells. Proc Natl Acad Sci U S A, 79: 7824-
7827, 1982.

Dang, C. V. c-Myc target genes involved in cell growth, apoptosis, and
metabolism. Mol Cell Biol, 19: 1-11, 1999.

Nau, M. M., Brooks, B. J ., Battey, J ., Sausville, E., Gazdar, A. F., Kirsch, I. R.,
McBride, 0. W., Bertness, V., Hollis, G. F ., and Minna, J. D. L-myc, a new myc-

related gene amplified and expressed in human small cell lung cancer. Nature,
318: 69-73, 1985.

Schwab, M., Varmus, H. E., Bishop, J. M., Grzeschik, K. H., Naylor, S. L.,
Sakaguchi, A. Y., Brodeur, G., and Trent, J. Chromosome localization in normal

human cells and neuroblastomas of a gene related to c-myc. Nature, 308: 288-
291, 1984.

Ingvarsson, S., Asker, C., Axelson, H., Klein, G., and Sumegi, J. Structure and
expression of B-myc, a new member of the myc gene family. Mol Cell Biol, 8:
3168-3174, 1988.

Sugiyama, A., Kume, A., Nemoto, K., Lee, S. Y., Asami, Y., Nemoto, F.,
Nishimura, S., and Kuchino, Y. Isolation and characterization of s-myc, a member
of the rat myc gene family. Proc Natl Acad Sci U S A, 86: 9144-9148, 1989.

Taub, R., Kirsch, I., Morton, C., Lenoir, G., Swan, D., Tronick, S., Aaronson, S.,
and Leder, P. Translocation of the c-myc gene into the immunoglobulin heavy

chain locus in human Burkitt lymphoma and murine plasmacytoma cells. Proc
Natl Acad Sci U S A, 79: 7837-7841, 1982.

Bentley, D. L. and Groudine, M. Novel promoter upstream of the human c-myc
gene and regulation of c-myc expression in B-cell lymphomas. Mol Cell Biol, 6:
3481-3489, 1986.

Ray, D., Meneceur, P., Tavitian, A., and Robert-Lezenes, J. Presence of a c-myc
transcript initiated in intron 1 in Friend erythroleukemia cells and in other marine
cell types with no evidence of c-myc gene rearrangement. Mol Cell Biol, 7: 940-
945, 1987.

Liao, D. J. and Dickson, R. B. c-Myc in breast cancer. Endocr Relat Cancer, 7:
143-164, 2000.

Hann, S. R., King, M. W., Bentley, D. L., Anderson, C. W., and Eisenman, R. N.
A non-AUG translational initiation in c-myc exon 1 generates an N-terminally

distinct protein whose synthesis is disrupted in Burkitt's lymphomas. Cell, 52:
185-195, 1988.

40

128.

129.

130.

131.

132.

133.

134.

135.

136.

137.

138.

139.

140.

Spotts, G. D., Patel, S. V., Xiao, Q., and Hann, S. R. Identification of
downstream-initiated c-Myc proteins which are dominant-negative inhibitors of
transactivation by full-length c-Myc proteins. Mol Cell Biol, 1 7: 1459-1468,
1997.

Dang, C. V. and Lee, W. M. Identification of the human c-myc protein nuclear
translocation signal. Mol Cell Biol, 8: 4048-4054, 1988.

Pelengaris, S., Khan, M., and Evan, G. c-MYC: more than just a matter of life and
death. Nat Rev Cancer, 2: 764-776, 2002.

Amati, B., Dalton, S., Brooks, M. W., Littlewood, T. D., Evan, G. I., and Land, H.
Transcriptional activation by the human c-Myc oncoprotein in yeast requires
interaction with Max. Nature, 359: 423-426, 1992.

Ayer, D. E., Kretzner, L., and Eisenman, R. N. Mad: a heterodimeric partner for
Max that antagonizes Myc transcriptional activity. Cell, 72: 211-222, 1993.

Kretzner, L., Blackwood, E. M., and Eisenman, R. N. Myc and Max proteins
possess distinct transcriptional activities. Nature, 359: 426-429, 1992.

La Rocca, S. A., Crouch, D. H., and Gillespie, D. A. c-Myc inhibits myogenic
differentiation and myoD expression by a mechanism which can be dissociated
from cell transformation. Oncogene, 9: 3499-3508, 1994.

Ryan, K. M. and Bimie, G. D. Cell-cycle progression is not essential for c-Myc to
block differentiation. Oncogene, 14: 2835-2843, 1997 .

Haggerty, T. J., Zeller, K. 1., Ostlrus, R. C., Wonsey, D. R., and Dang, C. v. A
strategy for identifying transcription factor binding sites reveals two classes of
genomic c-Myc target sites. Proc Natl Acad Sci U S A, 100: 5313-5318, 2003.

Nasi, S., Ciarapica, R., Jucker, R., Rosati, J ., and Soucek, L. Making decisions
through Myc. FEBS Lett, 490: 153-162, 2001.

Galaktionov, K., Chen, X., and Beach, D. Cdc25 cell-cycle phosphatase as a
target of c-myc. Nature, 382: 511-517, 1996.

Lasorella, A., Noseda, M., Beyna, M., Yokota, Y., and Iavarone, A. Id2 is a
retinoblastoma protein target and mediates signalling by Myc oncoproteins.
Nature, 407: 592-598, 2000.

Daksis, J. 1., Lu, R. Y., Facchini, L. M., Marhin, W. W., and Penn, L. J. Myc
induces cyclin D1 expression in the absence of de novo protein synthesis and

links mitogen-stimulated signal transduction to the cell cycle. Oncogene, 9: 3635-
3645, 1994.

41

141.

142.

143.

144.

145.

146.

147.

148.

149.

150.

151.

Philipp, A., Schneider, A., Vasrik, I., Finke, K., Xiong, Y., Beach, D., Alitalo, K.,
and Eilers, M. Repression of cyclin D1: a novel function of MY C. Mol Cell Biol,
14: 4032-4043, 1994.

Solomon, D. L., Philipp, A., Land, H., and Eilers, M. Expression of cyclin D1
mRNA is not uprcgulated by Myc in rat fibroblasts. Oncogene, 11: 1893-1897,
1 995.

Claassen, G. F. and Hann, S. R. A role for transcriptional repression of p21CIPl
by c-Myc in overcoming transforming growth factor beta -induced cell-cycle
arrest. Proc Natl Acad Sci U S A, 97: 9498-9503, 2000.

Gartel, A. L., Ye, X., Goufrnan, E., Shianov, P., Hay, N., Najmabadi, F., and
Tyner, A. L. Myc represses the p21(W AF 1/CIP1) promoter and interacts with
Spl/Sp3. Proc Natl Acad Sci U S A, 98: 4510-4515, 2001.

Seoane, J ., Le, H. V., and Massague, J. Myc suppression of the p21(Cipl) Cdk
inhibitor influences the outcome of the p53 response to DNA damage. Nature,
419: 729-734, 2002.

Wu, S., Cetinkaya, C., Munoz-Alonso, M. J ., von der Lehr, N., Bahram, F.,
Beuger, V., Eilers, M., Leon, J ., and Larsson, L. G. Myc represses differentiation-
induced p21CIPl expression via Miz-l -dependent interaction with the p21 core
promoter. Oncogene, 22: 351-360, 2003.

Mitchell, K. O. and El-Deiry, W. S. Overexpression of c-Myc inhibits

p21WAF l/CIPl expression and induces S-phase entry in 12-0-
tetradecanoylphorbol-l3-acetate (TPA)-sensitive human cancer cells. Cell Grth
Differ, 10: 223-230, 1999.

Yang, W., Sherr, J ., Wu, M., Arsura, M., FitzGerald, M., Suldan, 2., Kim, D. W.,
Hofrnann, C. S., Pianetti, S., Romieu-Mourez, R., Freedman, L. P., and
Sonenshein, G. E. Repression of transcription of the p27(Kip1) cyclin-dependent
kinase inhibitor gene by c-Myc. Oncogene, 20: 1688-1702, 2001.

Bems, K., Hijmans, E. M., and Bemards, R. Repression of c-Myc responsive
genes in cycling cells causes G1 arrest through reduction of cyclin E/CDK2
kinase activity. Oncogene, 15: 1347-1356, 1997.

Dubik, D., Dembinski, T. C., and Shiu, R. P. Stimulation of c-myc oncogene
expression associated with estrogen-induced proliferation of human breast cancer
cells. Cancer Res, 4 7: 6517-6521, 1987.

Dubik, D. and Shiu, R. P. Transcriptional regulation of c-myc oncogene
expression by estrogen in hormone-responsive human breast cancer cells. J Biol
Chem, 263: 12705-12708, 1988.

42

152.

153.

154.

155.

156.

157.

158.

159.

160.

161.

162.

Murphy, L. J ., Murphy, L. C., and Friesen, H. G. Estrogen induction of N-myc
and c-myc proto-oncogene expression in the rat uterus. Endocrinology, 120:
1882-1888, 1987.

van der Burg, B., van Selrn-Miltenburg, A. J ., de Laat, S. W., and van Zoelen, E.
J. Direct effects of estrogen on c-fos and c-myc protooncogene expression and

cellular proliferation in human breast cancer cells. Mol Cell Endocrinol, 64: 223-
228, 1989.

Dubik, D. and Shiu, R. P. Mechanism of estrogen activation of c-myc oncogene
expression. Oncogene, 7: 1587-1594, 1992.

Shang, Y. and Brown, M. Molecular determinants for the tissue specificity of
SERMs. Science, 295: 2465-2468, 2002.

Venditti, M., Iwasiow, B., Orr, F. W., and Shiu, R. P. C-myc gene expression
alone is sufficient to confer resistance to antiestrogen in human breast cancer
cells. Int J Cancer, 99: 35-42, 2002.

Watson, P. H., Pon, R. T., and Shiu, R. P. Inhibition of c-myc expression by
phosphorothioate antisense oligonucleotide identifies a critical role for c-myc in
the grth of human breast cancer. Cancer Res, 51 : 3996-4000, 1991.

Carroll, J. S., Swarbrick, A., Musgrove, E. A., and Sutherland, R. L. Mechanisms
of growth arrest by c-myc antisense oligonucleotides in MCF-7 breast cancer

cells: implications for the antiproliferative effects of antiestrogens. Cancer Res,
62: 3126-3131, 2002.

Clarke, R., Liu, M. C., Bouker, K. B., Gu, Z., Lee, R. Y., Zhu, Y., Skaar, T. C.,
Gomez, B., O'Brien, K., Wang, Y., and Hilakivi-Clarke, L. A. Antiestrogen

resistance in breast cancer and the role of estrogen receptor signaling. Oncogene,
22: 7316-7339, 2003.

Kasid, A., Lippman, M. E., Papageorge, A. G., Lowy, D. R., and Gelrnann, E. P.
Transfection of v-rasH DNA into MCF-7 human breast cancer cells bypasses
dependence on estrogen for tumorigenicity. Science, 228: 725-728, 1985.

Smith, L. M., Wise, S. C., Hendricks, D. T., Sabichi, A. L., Bos, T., Reddy, P.,
Brown, P. H., and Birrer, M. J. cJun overexpression in MCF-7 breast cancer cells

produces a tumorigenic, invasive and hormone resistant phenotype. Oncogene,
18: 6063-6070, 1999.

Johnston, S. R., Saccani-Jotti, G., Smith, I. E., Salter, J ., Newby, J ., Coppen, M.,
Ebbs, S. R., and Dowsett, M. Changes in estrogen receptor, progesterone receptor,

and p82 expression in tamoxifen-resistant human breast cancer. Cancer Res, 55:
3331-3338, 1995.

43

163.

164.

165.

166.

167.

168.

169.

Osborne, C. K. Mechanisms for tamoxifen resistance in breast cancer: possible
role of tamoxifen metabolism. J Steroid Biochem Mol Biol, 47: 83-89, 1993.

Wolf, D. M., Langan-Fahey, S. M., Parker, C. J ., McCague, R., and Jordan, V. C.
Investigation of the mechanism of tamoxifen-stimulated breast tumor growth with

nonisomerizable analogues of tamoxifen and metabolites. J Natl Cancer Inst, 85:
806-812, 1993.

Clarke, R., Leonessa, F., Welch, J. N., and Skaar, T. C. Cellular and molecular
pharmacology of antiestrogen action and resistance. Pharmacol Rev, 53: 25-71,
2001.

Dhillon, N. K. and Mudryj, M. Ectopic expression of cyclin B in estrogen
responsive cells abrogates antiestrogen mediated growth arrest. Oncogene, 21:
4626-4634, 2002.

Varma, H. and Conrad, S. E. Reversal of an antiestrogen-mediated cell cycle
arrest of MCF-7 cells by viral tumor antigens requires the retinoblastoma protein-
binding domain. Oncogene, 19: 4746-4753, 2000.

Wilcken, N. R., Prall, O. W., Musgrove, E. A., and Sutherland, R. L. Inducible
overexpression of cyclin D1 in breast cancer cells reverses the growth-inhibitory
effects of antiestrogens. Clin Cancer Res, 3: 849-854, 1997.

Akli, S., Zheng, P. J ., Multani, A. S., Wingate, H. F., Pathak, S., Zhang, N.,
Tucker, S. L., Chang, S., and Keyomarsi, K. Tumor-specific low molecular
weight forms of cyclin B induce genomic instability and resistance to p21, p27,
and antiestrogens in breast cancer. Cancer Res, 64: 3198-3208, 2004.

44

CHAPTER TWO

Regulation of Cell Cycle Progression by Estrogen, Antiestrogen and c-Myc
Induction in MCF-7 cells

Figure 1 and related contents in the text of this chapter were originally published in
Skildum A.J., Mukherjee S., and Conrad SE. 2001. The cyclin dependent kinase
inhibitor p21WAF1/Cm is an antiestrogen regulated inhibitor of Cdk4 in human breast
cancer cells. J Biol Chem. 277(7): 5145-52.

Figure 5C and 5D and related contents in the text of this chapter were ori 'nally
published in Mukherjee S., and Conrad SE. 2005. c-Myc suppresses p21W FUCIP‘
expression during estrogen signaling and antiestrogen resistance in human breast cancer
cells. J Biol Chem. Published online ahead of print, Manuscript: M502278200 (In
Press).

45

ABSTRACT
Forty percent of breast cancers are estrogen-dependent and sensitive to treatment with
antiestrogens. However, many tumors eventually become antiestrogen resistant. The
molecular mechanisms by which tumors progress to an antiestrogen-resistant phenotype
are poorly understood, and elucidating them is crucial for developing improved
therapeutics. Amplification of the c-myc gene and/or aberrant expression of its protein
product are common features of clinical breast cancer, and deregulated c-Myc expression
has been implicated in antiestrogen resistance. We used the MCF-7 cell line, which is an
estrogen receptor-positive human breast cancer cell line, to examine the role of c-Myc in
estrogen-stimulated proliferation of breast cancer cells and in antiestrogen-resistant cell
cycle progression. We first characterized the MCF-7 cell line used in our laboratory, and
confirmed that cell cycle progression and c-Myc expression in this subline are regulated
by estrogen and antiestrogens. We then used RNA interference to verify that c-Myc is
important for estrogen-stimulated proliferation of MCF-7 cells. Finally, we established
an independent system for ectopic c-Myc induction in MCF-7 cells, and corroborated
previous studies demonstrating that ectopic expression of c-Myc is sufficient to induce
cell cycle progression in the presence of antiestrogens. Together, these results have
independently validated the role of c-Myc in estrogen-stimulated proliferation of MCF-7

cells, and its ability to promote cell cycle progression in the presence of antiestrogens.

46

INTRODUCTION

Breast cancer is the most common cancer affecting women in the United States,
and is the second leading cause of cancer mortality, resulting in more than 43,000 deaths
each year (1). A majority of primary human breast cancers are estrogen receptor (ER)-
positive, and approximately 40% require estrogenic steroids such as 17B-estradiol (E2)
for proliferation (2). The key role of E2 in breast cancer proliferation has provided the
rationale for the development of antiestrogenic compounds, such as Tamoxifen and ICI
182,780 (ICI), for the treatment of breast cancers (3-5). Unlike chemotherapy, endocrine
therapy is well tolerated and has relatively few side effects. Unfortunately, a significant
proportion of patients with initially responsive tumors experience recurrence, indicating
the development of acquired antiestrogen resistance (6-8). As the prognosis for patients
with such tumors is generally poor, the development of resistance to hormonal therapy
represents a major obstacle in the treatment of breast cancer patients. The molecular
changes underlying the progression of breast cancer cells from an E2-dependent,
antiestrogen-sensitive to an E2-independent, antiestrogen-resistant phenotype, are poorly
characterized. Understanding the mechanisms by which E2 and antiestrogens regulate
proliferation of breast cancer cells is crucial, as this would provide insight into how
antiestrogen resistance might arise and suggest strategies to prevent or reverse its
development.

Various proteins have been identified as potential mediators of E2-induced
mitogenesis. One such target is the proto-oncogene c-Myc, which is rapidly induced
upon E2 treatment of MCF-7 cells and can impinge upon the cell cycle machinery (9-13).

A meta-analysis has established that at least 15% of breast cancer biopsies bear a c-myc

47

gene amplification, and in a recent study 70% of high-grade breast carcinomas showed
elevated c-Myc expression (14, 15). Amplification of c-myc is thought to be an
independent powerful prognosticator, particularly in steroid receptor—positive breast
cancer, where patients with tumors having amplified c-myc show a 46% increase in 5-
year recurrence rates when compared to patients with tumors having normal c-myc gene
copy numbers (16). Therefore, c-myc amplification and its deregulated expression may
play a significant role in the progression of tumors to an antiestrogen-resistant phenotype.
The goal of this study was to examine the role of c-Myc in E2-stimulated proliferation of
breast cancer cells and in antiestrogen-resistant cell cycle progression.

The effects of E2 and antiestrogen treatments on cell cycle progression have
primarily been investigated using in vitro models of human breast cancer. These models
consist of tumor-derived cell lines from patients with advanced breast cancer. In 1973,
the first successful attempt at this approach resulted in the development of an E2-
responsive, ER-positive breast cancer cell line at the Michigan Cancer Foundation (17-
19). The cell line was obtained from the pleural effusion of a patient with metastatic
breast cancer, and was designated the MCF-7 cell line. The MCF-7 cell line shows
aberrant expression of several cell cycle regulatory proteins; for example, the cyclin-
dependent kinase inhibitor p16 is absent, and cyclin E is constitutively expressed (20,
21). These cells proliferate in response to E2 in tissue culture media, as well as when
implanted into nude mice. Furthermore, tumors derived from MCF-7 cells in nude mice
are dependent upon E2 for growth, as ovariectomized mice do not form tumors without
E2 supplementation (22). Consequently, proliferation of the MCF-7 cell line is sensitive

to antiestrogens both in vitro and in vivo (23, 24). The MCF-7 cell line is now very

48

widely studied as a model of E2-dependent, antiestrogen-sensitive human breast cancer.
However, several decades of use in independent laboratories have resulted in the
evolution of distinct MCF-7 lineages. Differences have been documented in various
characteristics of the sublines including their ability to undergo DNA fragmentation,
tumorigenicity, proliferation rates, expression of ER and progesterone receptor, and
sensitivities to E2 and antiestrogens (25-28). One objective of this study was therefore to
independently verify pertinent characteristics of the MCF-7 cell line to be used in further
studies.

We characterized the regulation of cell cycle progression and c-Myc expression
by ICI and E2 in the MCF-7 cell line used in our laboratory. We then used RNA
interference to verify that c-Myc is important for E2-stimulated proliferation of these
MCF-7 cells. Finally, we established an independent system for ectopic c-Myc induction
in MCF-7 cells, and corroborated previous studies which demonstrated that ectopic
expression of c-Myc is sufficient to induce cell cycle progression of MCF-7 cells in the
presence of ICI. Together, these results have independently confirmed the role of c-Myc
in E2-stimulated proliferation in MCF-7, and its ability to promote cell cycle progression

of these cells in the presence of antiestrogens.

MATERIALS AND METHODS

Cell Lines and Cell culture. MCF-7 cells were obtained from the Lombardi Cancer

Center. Cells were maintained in improved modified Eagle’s medium (IMEM)

49

(BioFluids, Inc.) supplemented with 5% fetal bovine serum (FBS) (HyClone), 100
units/ml penicillin (Invitrogen), and 100 units/ml streptomycin (Invitrogen), and cultured
at 37°C with 5% CO2. When studying the effects of E2 and ICI, cells were cultured in
phenol red-free IMEM (BioFluids, Inc.) containing 5% charcoal-stripped fetal bovine
serum (CSS) (HyClone), and penicillin/streptomycin. For most experiments, cells were
plated in medium with PBS for 24 h, and pre-arrested for 48 h in CSS containing medium
with 10 nM ICI (Zeneca Pharmaceuticals). Cells were then treated with CSS containing
medium with either 10 nM E2 (Sigma) or 10 nM ICI and harvested at various time
points. For some experiments, cells were not pre-arrested with ICI in order to improve
transfection efficiency. In these experiments, cycling cells were directly treated with
medium containing either 100 nM ICI or 1 nM E2, and then harvested. The higher ICI
concentration was used to observe rapid arrest in cycling cells, and 1 nM E2 was used

since cells were not being released from an ICI arrest.

Plasmids. The pCEN-F3p65/Z1F3/Neo and pLH-ZlZ-I-pL plasmids were obtained from
Ariad Pharmaceuticals (29). The pJS plasmid, containing human c-myc cDNA (exons 2
and 3), was a gifi from Dr. S. Mai (30). The c-myc fragment was excised from pJS using
HindIII and BglII, subcloned into pLH-Z12-I-pL at the HindIII and EcoRI sites to
generate pLH-ZlZ-I-pL-Myc, and the orientation of the insert was confirmed by
restriction digestion and sequencing. The c-myc cDNA fragment obtained from pJ5 was

also used as a probe for Northern blot hybridizations.

50

Construction of MCF-7 Inducible Cell Lines. MCF-7/Myc stable transfectants were
generated from MCF-7 cells using the Argent TM Regulated Transcription Plasmid Kit
(Version 1) from Ariad Pharmaceuticals (29). MCF-7 cells were first stably transfected
with pCEN-F3p65/ZIF3/Neo using Lipofectin reagent (Invitrogen). Stable transfectants
were selected using Geneticin (Sigma) at 400 jig/ml, and subsequently maintained in 50
ug/ml Geneticin. Individual colonies were picked and cultured separately. They were
screened for expression of pCEN-F3p65/ZlF3/Neo using transient transfections with a
plasmid containing a secreted alkaline phosphatase reporter gene. Ms. Cunjie Zhang
carried out all the above steps. A stable derivative, giving low basal and high alkaline
phosphatase activity upon treatment with the small molecule inducer AP1510 (AP), was
then transfected with pLH-ZlZ-I-pL-Myc. Transfections with the pLH-ZlZ-I-pL vector
alone were also carried out to obtain vector control cell lines. Stable derivatives were
selected using Hygromycin (Invitrogen) at 35 ug/ml, and subsequently maintained in 10
jig/ml Hygromycin. Individual colonies were then picked and cultured separately. The
transfectants were screened by both Northern and Western blot analysis for induction of
c-myc mRNA and protein upon treatment with 300 nM AP. Selected clones were

thereafter maintained in medium containing Geneticin (50 ug/ml) and Hygromycin (10

Its/m1).

Northern Blotting. Cells were lysed, and total RNA was purified using Trizol reagent
(Invitrogen). Ten micrograms of RNA were electrophoresed on 1% forrnaldehyde-
agarose gels, transferred to nitrocellulose membranes (Schleicher and Schuell), and

hybridized with a 32P-labeled cDNA probe for c-myc.

51

Western Blotting. MCF-7 cells were lysed as described previously (31), and protein in
the cell lysates was quantitated using the Bradford protein assay (Bio-Rad). Proteins
(IOug) were resolved by 12% SDS-PAGE, transferred to polyvinylidene difluoride
membranes (Perkin Elmer), and probed with primary antibodies for c-Myc (clone 9E10,
ATCC) (32), ERor Ab17 (hybridoma supematent was a gift from Dr. R.J. Miksicek) (33),
or B-actin (clone AC-40, Sigma). After washing, membranes were incubated with
horseradish peroxidase-conjugated goat anti-mouse (American Qualex) secondary
antibodies. Bands were visualized using Super Signal West Pico Chemiluminescent

Substrate (Pierce).

Cell Cycle Analysis. Cells were trypsinized, washed in phosphate buffered saline (PBS),
suspended in PBS + 10% FBS, fixed with 80% cold ethanol and stored at -20°C. Prior to
analysis, cells were washed twice with PBS, and suspended in PBS containing 1 mg/ml
RNAase A, 0.2 mg/ml propidium iodide, 0.5 mM EDTA and 0.1% Triton X-100. Cells
were then analyzed for red fluorescence on a F ACSVantage flow cytometer, and cell

cycle was distribution determined using ModFitLT software.

Chromatin Immunoprecipitation (ChIP) Assays. ChIP assays were performed as
described previously (34). Briefly, MCF-7 cells were cultured for three days in medium
containing CSS, treated with 100 nM ICI or E2 for 45 minutes, and then cross-linked
with 1% formaldehyde for 10 minutes. Cell were lysed, sonicated to get approximately
600-800 bp fragments, and then chromatin was irnmunoprecipitated with antibodies

against ER (ERoc AblO, MS-315-P1, Neomarkers, Freemont, CA) or control mouse IgG

52

(so-2025, Santa Cruz Biotechnology). The cross-linking was reversed, DNA extracted,
and purified. PCR analysis was performed with both input chromatin and
immunoprecipitated samples, using primers specific for the c-myc promoter and GAPDH
exon 2. PCR products were separated by 2% Tris-borate-EDTA-agarose gel
electrophoresis, stained with ethidium bromide, and visualized with Kodak imaging

software.

Gene Silencing with small interfering RNAs (siRNAs). MCF-7 cells were cultured in
6-well plates containing 3 ml of medium with FBS. After 24 h, cells were transfected
with 100 nM each of c-myc and negative control siRNA duplexes (Ambion), using
siPORT TM Lipid Transfection Agent (Ambion), according to manufacturer’s
recommendations. Transfections were carried out for 4 h in serum free medium (SFM),
followed by treatment with CSS containing medium with 1 nM E2. Cells were harvested
after 48 h, and analyzed by flow cytometry or Western blotting. Cells maintained in
SFM for 4 h, and harvested after treatment with 100 nM ICI or 1 nM E2 for 48 h, served

as untransfected controls.

RESULTS
Cell cycle progression is regulated by ICI and E2 in MCF-7 cells. We characterized

regulation of cell cycle progression by E2 and ICI using the MCF-7 cell line available in

our laboratory. To verify E2-stimulated proliferation of this cell line, cells were plated in

53

60

 

4o-

30-

20-

Percent of cells in S phase

 

 

O
O)

12 18 24 3O 36 48
Duration of treatment (h)

 

Percent of cells in S phase

 

 

0 6 12 18 24 30 36 48
Duration of treatment (h)

Figure 1: Cell cycle progression is regulated by ICI and E2 in MCF-7 cells. MCF-7
cells were either pre-arrested with medium containing CSS with 10 nM ICI (A) or treated
with medium containing CSS with 10 nM E (B) for 48 h. The cells were then either
released with 10 nM E (A) or arrested with 10 nM ICI (B), and harvested at 6 h intervals
for 36 h and a 48 h time point was also included. Cell cycle phase distribution was
determined by propidium iodide staining of DNA and analysis by flow cytometry. The
average percentage of cells in S phase for each time point is shown +/- standard
deviation.

medium with FBS for 24 h. Cells were then pre-treated with CSS containing medium
with 10 nM ICI for 48 h to block ER signaling. This medium was then replaced with
CSS containing medium with 10 nM E2, and cells were harvested every 6 h for 36 h. A
48 h time point was also included. Cell cycle phase distribution was then determined by
propidium iodide staining of DNA and flow cytometry as described in “Materials and
Methods”. The percent of cells in S phase at each time point is shown in Figure 1A.
Cells that were pre-arrested in the G1 phase of the cell cycle by ICI, were slowly released
fiom the arrest, and started entering the S phase by 18 h after addition of E2. A majority
of the population was in the S phase by 24 h; thereafter, the percent of cells in S phase
decreased by 30-36 h as the cells started entering G2/M. The percent of cells in S phase
rose once again at 48 h, indicating passage through the second cell cycle.

A converse approach was used to confirm inhibition of cell cycle progression by
ICI. Cells were plated in medium with F BS as described above, and after 24 h they were
treated with E2 for 48 h to generate a proliferating, asynchronous population. E2 was
then removed, medium with ICI was added, and samples Were harvested every 6 h and
analyzed by flow cytometry. As shown in Figure 1B, cells growing in E2 are gradually
arrested by ICI starting at 18 h and reach a minimum of 5% in S phase by 36-48 h.
Together, these time course experiments showed that proliferation of the MCF-7 cell line
used is regulated by E2 and ICI, and defined the experimental conditions for growth

arrest with ICI and release from arrest with E2.

c-Myc expression is regulated by E2 and ICI in MCF-7 cells. To verify regulation of

c-Myc expression by E2, cells were plated in medium with FBS for 24 h, and then treated

55

with CSS containing medium alone or with ICI or E2 for 56 h. For one sample, cells
were pre-arrested with ICI for 48 h, and then released from growth arrest by removal of
ICI and treatment with E2 for 8 h. The levels of c-Myc were then determined by Western
blotting (Figure 2A). Levels of c-Myc were low in the absence of E2, and increased upon
treatment with E2. Thus, it was established that c-Myc expression is regulated by E2 in
the MCF-7 cell line used. In the asynchronous, cycling population generated by
treatment with E2 for 56 hours, the levels of c-Myc were higher than in the population
pre-arrested with ICI and released for 8 h with E2 (E2* in Figure 2A). This was possibly
because in the pre-arrested cells, ICI decreased ER levels, and they had not completely
recovered from the ICI arrest by 8 h (35, 36).

To further characterize c-myc regulation by E2 and ICI, ChIP assays were carried
out to test for the presence of ER at the c-myc promoter under these conditions. Figure
2B shows the results obtained upon E2 treatment of MCF-7 cells. Chromatin
Immunoprecipitation was carried out using anti-ER or control IgG antibodies, followed
by PCR analysis using primers for the c-myc promoter. This demonstrated specific
binding of ER at the c-myc promoter in the presence of E2 (Lane 4 versus Lane 5). Lanes
1 to 3 show lO-fold serial dilutions of input chromatin. A control in which no DNA was
put into the PCR reaction showed no product, and confirmed that there was no
contamination with external c-myc DNA (Lane 6). PCR with primers specific for
GAPDH served as a negative control, and indicated that ER binds specifically to the c-
myc promoter DNA. Subsequently, the presence of ER at the c-myc promoter was

compared in the presence of E2 versus ICI. As shown in Figure 2C, immunoprecipitation

56

CSS ICI 52* E2
1’ - ' _...-_. ‘_ h I -I—-:_-7 i
a. -w a J C'Myc

1
I

"'" .._. —- —-— Actin

Input

/l ER IgG No

0.001 0.01 0.1 IP IP DNA

 

 

 

 

 

 

 

 

 

 

 

 

 

“7“”. __ .fl. c-myc
ET? T 3’ Mi 6" - “ ‘Q’ 7 ”I _ “ E GAPDH
1 2 3 4 5 6
C.
Input
/1 ER IgG N0
0.001 0.01 0.1 IP “3 DNA
’I....i—---2“"i -- IE
. 2”" .n, '1" "i “m?" I . j
.. -- +- 1

 

Figure 2': Regulation of c-Myc by E2 and ICI in MCF-7 cells. A) Cells were treated
for 56 h with either medium containing CSS alone or with 10 nM ICI, or 10 nM E2; or
pre-arrested with 10 nM ICI for 48 h, and released from grth arrest by removal of ICI
and treatment with 10 nM E2 for 8 h (represented as B“). Cells were then harvested and
c-Myc levels determined by Western blotting. Actin served as a loading control. B) and
C) MCF-7 cells were cultured for 3 days in medium containing CSS, then treated with
E2/ICI for 45 minutes, and cross-linked with formaldehyde for 10 minutes. Cells were
lysed, sonicated, and chromatin immunoprecipitated with antibodies. against ER or
control IgG. The cross-linking was reversed, DNA extracted, purified and analyzed by
PCR using primers specific for the c-myc promoter and GAPDH exon 2. Lanes 1 to 3
demonstrate 10-fold serial dilutions of input chromatin. The experiments shown in B)

and C) were each carried out once. These experiments were performed in collaboration
with Ms. Emily Flynn, and Mr. Yi Zheng.

57

with ER or control antibodies demonstrated that ER specifically binds to the c-myc
promoter in the presence of E2 but not ICI.

These results corroborate recent studies, which used ChIP assays to demonstrate
that E2 treatment leads to ER binding and co-activator recruitment at the c-myc promoter,
resulting in the rapid induction of c-Myc (34, 37-39). Our results extended these studies
by demonstrating that while ER is at the c-myc promoter in the presence of E2, it is not
recruited in the presence of ICI. Together, these studies confirmed that c-Myc expression
is regulated by ICI and E2 in the MCF-7 cell line, and suggested that altered ER

recruitment to the c-myc promoter, may account for this regulatory effect.

c-Myc expression is important for E2-stimulated proliferation of MCF-7 cells.
Previous studies using c-myc antisense oligonucleotides, and studies published this year
using c-myc small interfering RNAs (siRNA), have demonstrated that decreasing c-Myc
levels reduces proliferation of E2-treated MCF-7 cells, indicating that c-Myc is important
for E2-stimulated proliferation (40-43). We independently validated the results obtained
with antisense oligonucleotides, by using c-myc siRNA to reduce c-Myc levels in our
MCF-7 cell line. We transfected cells for 4 h with 100nM of either c-myc or negative
control siRNA, and then treated them with E2 for 48 h. Thereafter, cells were harvested
and analyzed by Western blotting or flow cytometry. c-myc siRNA, but not control
siRNA, decreased c-Myc protein in E2-treated cells to levels similar to those seen in ICI-
treated cells, indicating that the above conditions can efficiently reduce c-Myc levels in
MCF-7 cells (Figure 3A). In addition, siRNA did not cause a nonspecific

downregulation of gene expression, as determined by the B-actin control. As shown in

58

ICI 52 E2 E2
+msi +csi

 

.3654”- Actin

H _

 

100

0 cells
\1 00 (O
O O O
l l 1

”"602

Percentage
N (a) A 01
O O O O

1 l l l

 

 

 

 

S
Cell cycle phase

Figure 3: Effect of c-myc siRNA on c-Myc expression and cell cycle progression in
E2-treated MCF-7 cells. Cycling MCF—7 cells were transfected with 100 nM c-myc
siRNA (msi) or control siRNA (csi) duplexes as described in “Materials and Methods”,
and then treated with E2 for 48 h. A) Cells were harvested and cell lysates were analyzed
by Western blotting. Levels of c-Myc and actin were compared to those seen in
untransfected cells treated with ICI or E2. B) Cells were harvested and cell cycle
distribution determined by flow cytometry. The average percentage of cells in each
phase for each treatment is shown +/- standard deviation. White bars, ICI. Hatched bars,
E2+msi. Gray bars, E2. Black bars, E2+csi.

59

Figure 3B, c-myc siRNA decreased the percentage of cells in S phase approximately two
fold compared to control siRNA or E2-treated cells. This decrease in percent S phase
was not as much as that seen in ICI, suggesting either that c-Myc was not downregulated
in the proliferating cells, or that ICI targets additional proteins which regulate
proliferation. These results are in agreement with those obtained in previous studies, in
which DNA synthesis was decreased by approximately 30% upon 16 h of treatment with
c-myc antisense oligonucleotides, or by 50% after 3 days of treatment with c-myc siRNA
(41, 42). They therefore demonstrate that c-myc siRNA can effectively decrease the
levels of c-Myc in E2-treated cells to those seen in ICI-treated cells, and confirmed that

c-Myc is important for E2-stimulated proliferation of MCF-7 cells.

Generation of stably transfected cell lines in which c-myc is expressed from an
inducible promoter. To determine the effects of c-myc expression on proliferation of
MCF-7 cells in the presence of ICI, a stably transfected cell line was constructed in which
ectopic c-myc expression could be stringently controlled. Proliferation could therefore be
directly compared in the presence or absence of c-myc, in the same genetic background.
A novel gene regulation system obtained from ARIAD pharmaceuticals was used (29).
The system is based on small molecule regulated protein dimerization and consists of two
plasmids and a dimerizer AP1510 (AP) (Figure 4). The first plasmid (pCEN-
F3p65/Z1F3/Neo) has a neomycin resistance gene and encodes two distinct fusion
proteins, one containing a DNA—binding domain and the other a transcriptional activation
domain. Each of these domains is fused to a cellular protein (FKBP) that interacts with a

small molecule dimerizer (AP). In the absence of the dimerizer, the two fusion proteins

60

1. Stable transfection of the transcription factor plasmid (pCEN—F3p65/Z1F3lNeo)
3xFKBP l p65 H ZFHD1 l 3xFKBP

Activation domain DNA binding Domain

sip e

2. Stable transfection of target plasmid (pLH-Z12-l-pL-myc)

2‘2 e ret-

——-—I 12xZFHD1 m

l” AP1510

 

 

 

 

3. Add Dimerizer

 

 

—-—l 12mm t—--—-— '

Figure 4: Construction of MCF-7/Myc stably transfected cell lines. This is a
schematic of the regulatory system used to induce expression of c-myc in MCF-7/Myc
stable transfectants. MCF-7 cells were first stably transfected with pCEN—
F 3p65/ZlF3/Neo. Ms. Cunjie Zhang carried out this transfection. Expression from this
plasmid is driven by a human cytomegalovirus enhancer/promoter (CMV). It encodes a
neomycin selectable marker (Neo) and two fusion proteins, one containing a DNA—
binding domain (ZFHDl) and the other a transcriptional activation domain (derived from
the carboxy terminal of NF-kB p65 protein). Each of these domains is fused to three
tandemly repeated copies of human FKBP12 (FKBP), which interacts with the small
molecule dimerizer (AP). In the second step, cells containing pCEN-F3p65/ZlF3/Neo
were transfected with the target plasmid (pLH-ZlZ-I-pL-myc), which contains a
hygromycin selectable gene (Hygro), a minimal interleukin-2 gene promoter (1L2), and
12 copies of a DNA sequence recognized by ZFHDl (12xZFHD1). c-myc was cloned
into the polylinker region of the target plasmid. In the absence of AP, the two fusion
proteins have no affinity for one another, but in the presence of the dimerizer they
interact to form an active transcription factor, which binds to 12xZFHDl, and results in
transcription of c-myc. MCF-7/Myc cell lines can be screened for c-Myc induction after
treatment with AP.

61

have no affinity for one another, but in the presence of the dimerizer they interact to form
an active transcription factor. The second plasmid, pLH-Z12-I-pL, contains a
hygromycin resistance gene, and a polylinker that is used to clone the gene of interest (c-
myc). The polylinker lies downstream of a promoter containing DNA elements that are
recognized by the DNA binding domain encoded by pCEN-F3p65/Z1F3/Neo.
Transfections were carried out as described in “Material and Methods”. Thirty-
four individual transfectants were obtained, expanded into cell lines and assayed by
Northern Blotting for their ability to express ectopic c-myc mRNA in response to AP.
Cells were plated in medium with FBS, and after 4 days were treated with ICI containing
medium with or without AP. After 24 h, the cells were harvested, and c-myc mRNA
levels were analyzed by Northern blotting. Representative results for four e—myc
transfectants and one vector control cell line are shown in Figure 5A. c-myc mRNA
levels were low in the ICI-treated cells, thus establishing that the endogenous c-myc
mRNA levels were decreased by ICI, and that expression of the ectopic c-myc was not
leaky. In addition, vector control cells did not express the c-myc in response to AP,
demonstrating that treatment with AP does not induce endogenous c-myc. Any c-myc
mRNA seen in the presence of AP could therefore be attributed mainly to the expression
of ectopic c-myc. Several independently derived transfectants, such as MCF-7/Mycl8
and MCF-7/Myc31 (Figure 5A), showed induction of c-myc in ICI+AP-treated cells and
low levels of c-myc in ICI-treated cells, and were selected for further studies. Promising
clones selected by Northern Blot analysis were then screened by Western Blotting to

confirm induction of ectopic c-Myc protein by AP (data not shown and Figure 5C).

62

Figure 5: Characterization of cell lines with inducible c-Myc expression. Stable
MCF-7 derivatives were established as described in “Materials and Methods”. A) The
indicated stable transfectants were cultured in medium with FBS for 4 days, and then
treated with medium containing CSS with 10 nM ICI or 10 nM ICI + 300 nM AP. After
24 h, the cells were harvested, and c-myc mRNA levels were analyzed by Northern
blotting. B) The specified cell lines were treated with medium containing CSS with ICI
or E2 for 48 h, and then harvested. Cell lysates were analyzed by Western blotting for
ER levels. Actin served as an internal loading control. C) The indicated stable
transfectants were pre-arrested with medium containing CSS with ICI for 48 h, then
treated with medium containing CSS with ICI, ICI + AP (300 nM) or E2 (10 nM), and
harvested after 24 h. Cells lysates were subjected to Western blotting for c-Myc. Actin
served as a loading control. D) The cells treated as described in C) were harvested
concurrently with those used for Western blotting, and the cell cycle phase distribution
was determined by flow cytometry. The average percentage of cells in S phase for each
treatment is shown +/- standard deviation. Gray bars, ICI-treated cells. Black bars,
ICI+AP-treated cells. White bars, E2-treated cells.

63

A.

Myc18 Myc 22 Myc 28 Myc 31 Vector
- + f + - + - + - + AP

T —-..:

.i ”a .- . .19 c-myc

MCF-7 Myc/33 Vector
ICI E2 ICI E2 ICI E2

- ‘ l? a; Pew-H's. “1.13:7."va -u: ‘1. .

”v -‘ 9'- g g- , ER
..._ .. e . 1
habit—2*“;- .fl-d—a-‘efl4

 

 

1:32-11 1J3. »_. -~- A
L —-—' .. M '1' — ’r '1

Actin

 

C.

Myc18 Myc31 Myc33 Myc41 Vector
ICI E2 ICI E2 ICI E2 ICI E2 ICI E2

4;- 'v_v;. ngfi'pi‘ Actin

 

 

Percent of cells in S phase
0)
O

 

 

 

Myc18 My031 Myc33 Myc41 Vector
Stable Transfectants

64

Thus, stable transfectants were established, in which ectopic c-Myc levels can be tightly

regulated, and are induced by AP to levels similar to those seen in the presence of E2.

Characterization of cell lines with inducible c-Myc expression. To address the
possibility that the cell lines selected following transfection might have altered levels of
ER relative to the parental MCF-7 cell lines, cycling MCF-7, MCF-7fMyc33 and vector
control cells were treated with ICI or E2 for 48 h, and cell lysates were analyzed by
Western blotting. ER levels were downregulated in ICI-treated cells as compared to E2-
treated cells, and the levels observed was similar in all cell lines (Figure 5B).

Previous studies have shown that ectopic expression of c-Myc is sufficient to
induce cell cycle progression of MCF-7 cells in the presence of ICI (44, 45). To confirm
this result, both c-Myc protein expression and cell cycle progression were characterized
in several independently derived transfectants. Cells were pre-treated with ICI for 48 h to
block ER activity and cell proliferation. They were then treated with medium containing
ICI, ICI+AP or E2 for 24 h, harvested, and analyzed by flow cytometry and Western
blotting. c-Myc protein levels were low in the presence of ICI and increased upon E2
treatment in all transfectants (Figure 5C), indicating that endogenous c-Myc was
regulated as expected. Expression of ectopic c—Myc was tightly regulated, and could be
induced by AP in the presence of ICI to levels comparable to those seen in E2-treated
cells. AP treatment did not induce c-Myc expression in a vector control cell line,
demonstrating that treatment with AP only induces expression of ectopic c-Myc and not

endogenous c-Myc.

65

Cell cycle distribution was examined by flow cytometry, and the percentage of
cells in S phase after 24 h of treatment is shown in Figure 5D. In each transfectant, the
percentage of cells in S phase was higher after E2 than ICI treatment. Upon treatment
with ICI+AP, the percentage of cells in S phase was higher than in ICI but lower than in
E2 treatments. This increase in the percentage of cells in S phase was specific to c-Myc
expression since it was not seen in vector control cells treated with ICI+AP. Thus, c-Myc
expression alone is sufficient to promote cell cycle progression in ICI-treated
transfectants, but not as well as E2 in most cases. The cell cycle distribution differences
between the various transfected cell lines might be due to the fact that only one time point
was examined, and the clonal cell lines may have progressed through the cell cycle at
somewhat different rates. The MCF-7/Myc33 and MCF-7/Myc3l cell lines were selected
for further study since they showed tight regulation of c-Myc and significant increases in

S phase in response to AP treatment.

Induction of c-Myc in the presence of ICI causes cell cycle progression with kinetics
similar to those seen in E2-treated cells. Time course experiments were carried out to
allow a detailed comparison of the kinetics of cell cycle progression in E2 and ICI+AP-
treated cells. MCF-7/Myc33 cells were plated in medium with FBS. They were then
pre-arrested with ICI for 48 h, treated with ICI, ICI+AP or E2, and harvested at 6 h
intervals for analysis by flow cytometry and Western blotting. The distribution of cells in
different phases of the cell cycle was determined, and the data for S phase is shown in
Figure 6A. Fewer than 5% of ICI-treated cells were in S phase at 0 h, and no significant

changes were seen throughout the time course. Upon treatment with ICI+AP the

66

 

50-

40«

302

202

10-

Percent of cells in S phase

 

 

 

 

 

Duration of treatment (h)

’ ICI+AP ‘ 52 lCl

A x

0 61218243036 61218 243036 61218243036 Tlrm(h)
.,.~.iflfim ‘l: '11: "II-H V .,,_ “'0‘ "1"

q
.*" C--- ' _ __
jab-1 _. , -OCCQQOq-g “So-Myc

- mi-lfil- a: ' -.r-—vv2-—éwm 2% .. .. .m

_ l —o w— ' l_.

Figure 6: Induction of ectopic c-Myc and cell cycle kinetics in ICI-treated MCF-7
cells. MCF-7/Myc33 cells were pre-arrested with ICI for 48 h, and then treated with ICI,
ICI+AP, or E2. Cells were harvested every 6 h for 36 h. A) Cell cycle analysis was
performed as described in “Materials and Methods”. The average percentage of cells in S
phase for each time point is shown +/- standard deviation. A, E2-treated cells. I,
ICI+AP-treated cells. 0, ICI-treated cells. B) Cell lysates were analyzed by Western
blotting for c-Myc. Actin served as a loading control.

67

percentage of cells in S phase was higher than in ICI but lower than in E2 treatments, as
seen previously (see Figure 5B). However, cell cycle phase kinetics for both ICI+AP-
and E2-treated cells were similar over the duration of this experiment. Cells in both
treatments began to enter S phase by 18 h, and substantial increases were seen between
18 and 24 h. The percentage of ICI+AP- or E2-treated cells in G2/M increased between
24 - 30 h (data not shown). The above analysis was also performed using the MCF-
7/Myc31 transfectant, which showed similar kinetics of S phase progression in ICI+AP-
and E2-treated cells (data not shown). As shown in Figure 6B, c-Myc protein levels were
low in the presence of ICI. ICI+AP treatment caused an increase in c-Myc protein within
6 h to levels sirrrilar to those seen in E2-treated cells. In this experiment, ectopic c-Myc
levels were decreased later in the time course, however this was not consistently observed
in other experiments. In a previous experiment (Figure 5B), at 24 h the levels of c-Myc
in ICI+AP- and E2- treated cells were similar, and the percentage of MCF-7/Myc cells in
S phase in both ICI+AP- and E2-treated cells were similar to those seen in this
experiment. These results agree with previous studies, which showed that c-Myc

induction is sufficient for cell cycle progression in the presence of ICI (44, 45).

DISCUSSION

Several proteins have been identified as potential mediators of E2-induced
mitogenesis in breast cancer cells, and aberrant expression of key targets has been shown

to promote cell cycle progression in the absence of E2 or the presence of antiestrogens.

68

A number of these E2 targets are cell cycle proteins, while others are gene products that
can in turn target cell cycle regulators (44-50). We chose to study the proto-oncogene
product c-Myc, which is aberrantly expressed in many breast tumors, since it is a target
of E2 in breast cancer cells, is required for E2-stimulated proliferation, and can impinge
upon the cell cycle machinery (9-15, 40, 41).

We carried out our studies in the MCF-7 cell line, which serves as a model for
E2-dependent and antiestrogen-sensitive breast cancer. Although other E2-responsive
human breast cancer cell lines have been established, including the T47D, ZR-75-1 and
CAMA-l cell lines, the MCF-7 cell line remains one of the most extensively studied in
vitro model systems (18, 51-54). MCF-7 cells have been distributed to laboratories all
over the world and have been continually passaged, which has led to the evolution of
distinct MCF-7 lineages (25). Therefore, before establishing stable MCF-7 derivatives to
study the role of c-Myc in E2 signaling and antiestrogen resistance, we characterized the
MCF-7 cell line available in our laboratory. Previous studies in our lab had demonstrated
that DNA synthesis in MCF-7 cells was stimulated by E2 and inhibited by ICI after 43 h
treatments with ICI/E2 followed by a 5 h labeling with BrdU (55). We extended these
studies by using flow cytometry to confirm that the MCF-7 cell line was responsive to E2
treatment and remained sensitive to ICI, and studied the kinetics of cell cycle progression
over an extensive 48 h time course (Figure 1). These studies also established conditions
wherein cells could be synchronized and then released fi'om arrest. Subsequently, we
verified that c-Myc expression was regulated by ICI and E2 in these MCF-7 cells (Figure

2A). We also used RNA interference to effectively decrease c-Myc levels in E2-treated

69

MCF-7 cells, and confirmed that c-Myc is important for E2-stimulated proliferation of
these cells (Figure 3).

We then examined whether ER was present at c-myc promoter in ICI- and E2-
treated MCF-7 cells (Figure 2 B, 2C). Previous studies using transient transfections with
c-myc promoter-reporter gene constructs have shown that 116 bp, including the TATA
box and upstream regions of the P2 promoter, are necessary for E2-regulated reporter
gene activity. An ERE half-site, and a GC-rich region (Sp-1 or Sp-l-like factor binding
site), have been mapped to this E2-responsive region of the c-myc promoter. It has been
proposed that the weak interaction between the E2-ER complexes at ERE half-sites is
stabilized by interaction with an adj acently bound Sp-l or Sp-l-like factor, thus leading
to ER-dependent c-myc expression (56). Recent studies using Chromatin
immunoprecipitation (ChIP) assays substantiated the above results by demonstrating that
E2 treatment leads to ER binding and co-activator recruitment at the c-myc promoter,
resulting in the rapid induction of c-Myc (34, 37-39). Our results confirmed and
extended the above studies by demonstrating that while ER is at the c-myc promoter in
the presence of E2, it is not recruited upon ICI treatment. This suggested that altered ER
recruitment to the c-myc promoter might account for the regulatory effects of E2 and ICI.
ICI can act by multiple mechanisms including impaired ER dimerization, increased
receptor degradation, and disrupted nuclear localization of ER, and all of these could
contribute to the lack of ER recruitment at the c-myc promoter in the presence of ICI (35,
36,57)

Finally, we established stable MCF-7 derivatives in which ectopic c-Myc

expression could be tightly regulated. Using these derivatives, we confirmed previous

70

reports that expression of c-Myc at levels comparable to those induced by E2 is sufficient
to promote cell cycle progression in the presence of ICI (Figures 5-6) (44, 45). We see
variability in our experiments with regard to the responsiveness of cells to E2, even
within the same stably transfected cell line. Release from ICI-arrest with E2 treatment
results in anywhere between 25 — 55% of the cells entering S phase at 24 h, and this
could in part be due to variation in the amount of other mitogens present in the batches of
CSS used for treatment. Treatment with ICI shows less variability, and regularly results
in decreases in the percentage of cells in S phase to approximately 5 - 15%. This is
possibly because ICI not only impairs ER dimerization, but also increases receptor
degradation (35, 36). Treatment of cells with ICI+AP, shows good reproducibility and
typically results in 20 - 25% of cells entering S phase at 24 h. However, inducing c-Myc
expression alone in the presence of ICI is usually not sufficient to restore cell cycle
progression to levels equivalent to those induced by E2. It is therefore likely that ICI
affects multiple targets that together with c-Myc regulate proliferation.

Our data corroborate previous work using an independent system for c-Myc
induction in human breast cancer cells. Of the two previous studies, one demonstrated
that c-Myc expression is sufficient for long term (8 day) proliferation in the presence of
ICI (45), while the other study focused on progression through one cell cycle (44). In the
latter study, both induction of c-Myc and E2 treatment of stable MCF-7/Myc
transfectants resulted in cells progressing through the cell cycle faster than similarly
treated MCF-7/vector control transfectants. This was due to the leaky expression of c-
Myc in that system, which affected studies relating to cell cycle kinetics. Since our

system offered a means for stringent control of ectopic c-Myc expression, we were able

71

to demonstrate that induction of c-Myc in the presence of ICI causes cell cycle
progression with kinetics similar to those seen in E2-treated cells.

In conclusion, we have characterized cell cycle regulation and c-Myc expression
in the MCF-7 cell line, and established a system that enables stringent control of ectopic
c-Myc expression in this cell line. In addition, we have independently validated the role
of c-Myc in E2-stimulated proliferation of MCF-7 cells, and its ability to promote cell

cycle progression of these cells in the presence of antiestrogens.

72

REFERENCES

Jemal, A., Clegg, L. X., Ward, E., Ries, L. A., Wu, X., Jamison, P. M., Wingo, P.
A., Howe, H. L., Anderson, R. N., and Edwards, B. K. Annual report to the nation
on the status of cancer, 1975-2001, with a special feature regarding survival.
Cancer, 101: 3-27, 2004.

Rutqvist, L. E., Cedermark, B., Glas, U., Johansson, H., Nordenskjold, B., Skoog,
L., Somell, A., Theve, T., Friberg, S., and Askergren, J. The Stockhohn trial on
adjuvant tamoxifen in early breast cancer. Correlation between estrogen receptor
level and treatment effect. Breast Cancer Res Treat, 10: 255-266, 1987.

Cole, M. P., Jones, C. T., and Todd, 1. D. A new anti-oestrogenic agent in late
breast cancer. An early clinical appraisal of ICI46474. Br J Cancer, 25 : 270-275,
1971.

DeFriend, D. J ., Anderson, E., Bell, J ., Wilks, D. P., West, C. M., Manse], R. E.,
and Howell, A. Effects of 4-hydroxytamoxifen and a novel pure antioestrogen

(ICI 182780) on the clonogenic growth of human breast cancer cells in vitro. Br J
Cancer, 70: 204-211, 1994.

DeFriend, D. J ., Howell, A., Nicholson, R. I., Anderson, E., Dowsett, M., Manse],
R. E., Blarney, R. W., Bundred, N. J ., Robertson, J. F., Saunders, C., and et a1.
Investigation of a new pure antiestrogen (ICI 182780) in women with primary
breast cancer. Cancer Res, 54: 408-414, 1994.

Systemic treatment of early breast cancer by hormonal, cytotoxic, or immune
therapy. 133 randomised trials involving 31,000 recurrences and 24,000 deaths
among 75,000 women. Early Breast Cancer Trialists' Collaborative Group.
Lancet, 339: 1-15, 1992.

Tamoxifen for early breast cancer: an overview of the randomised trials. Early
Breast Cancer Trialists' Collaborative Group. Lancet, 351: 1451-1467, 1998.

Wolf, D. M. and Jordan, V. C. William L. McGuire Memorial Symposium. Drug
resistance to tamoxifen during breast cancer therapy. Breast Cancer Res Treat, 27:
27-40, 1993.

Dubik, D., Dembinski, T. C., and Shiu, R. P. Stimulation of c-myc oncogene
expression associated with estrogen-induced proliferation of human breast cancer
cells. Cancer Res, 47: 6517-6521, 1987 .

73

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

Dubik, D. and Shiu, R. P. Transcriptional regulation of c-myc oncogene
expression by estrogen in hormone-responsive human breast cancer cells. J Biol
Chem, 263: 12705-12708, 1988.

van der Burg, B., van Selrn-Miltenburg, A. J ., de Laat, S. W., and van Zoelen, E.
J. Direct effects of estrogen on c-fos and c-myc protooncogene expression and

cellular proliferation in human breast cancer cells. Mo] Cell Endocrinol, 64 : 223-
228, 1989.

Dang, C. V. c-Myc target genes involved in cell growth, apoptosis, and
metabolism. Mol Cell Biol, 19: 1-11, 1999.

Nasi, S., Ciarapica, R., Jucker, R., Rosati, J ., and Soucek, L. Making decisions
through Myc. FEBS Lett, 490: 153-162, 2001.

Blancato, J ., Singh, B., Liu, A., Liao, D. J ., and Dickson, R. B. Correlation of
amplification and overexpression of the c-myc oncogene in high-grade breast

cancer: FISH, in situ hybridisation and irnmunohistochenrical analyses. Br J
Cancer, 90: 1612-1619, 2004.

Deming, S. L., Nass, S. J ., Dickson, R. B., and Trock, B. J. C-myc amplification

in breast cancer: a meta-analysis of its occurrence and prognostic relevance. Br J
Cancer, 83: 1688-1695, 2000.

Bems, E. M., Klijn, J. G., van Putten, W. L., van Staveren, I. L., Portengen, H.,
and F oekens, J. A. c-myc amplification is a better prognostic factor than
HER2/neu amplification in primary breast cancer. Cancer Res, 52: 1107-1113,
1992.

Brooks, S. C., Locke, E. R., and Soule, H. D. Estrogen receptor in a human cell
line (MCF-7) fiom breast carcinoma. J Biol Chem, 248: 6251-6253, 1973.

Lippman, M., Bolan, G., and Huff, K. The effects of estrogens and antiestrogens
on hormone-responsive human breast cancer in long-term tissue culture. Cancer
Res, 36: 4595-4601, 1976.

Soule, H. D., Vazguez, J ., Long, A., Albert, S., and Brennan, M. A human cell
line from a pleural effusion derived fiom a breast carcinoma. J Natl Cancer Inst,
51: 1409-1416, 1973.

Craig, C., Kim, M., Ohri, E., Wersto, R., Katayose, D., Li, Z., Choi, Y. H.,
Mudahar, B., Srivastava, S., Seth, P., and Cowan, K. Effects of adenovirus-
mediated p16lNK4A expression on cell cycle arrest are determined by

endogenous p16 and Rb status in human cancer cells. Oncogene, 16: 265-272,
1998.

74

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

Keyomarsi, K. and Pardee, A. B. Redundant cyclin overexpression and gene
amplification in breast cancer cells. Proc Natl Acad Sci U S A, 90: 1112-1116,
1 993.

Brunner, N., Bronzert, D., Vindelov, L. L., Rygaard, K., Spang-Thomsen, M., and
Lippman, M. B. Effect on growth and cell cycle kinetics of estradiol and
tamoxifen on MCF-7 human breast cancer cells grown in vitro and in nude mice.
Cancer Res, 49: 1515-1520, 1989.

Sutherland, R. L., Green, M. D., Hall, R. E., Reddel, R. R., and Taylor, I. W.
Tamoxifen induces accumulation of MCF 7 human mammary carcinoma cells in
the GO/Gl phase of the cell cycle. Eur J Cancer Clin Oncol, 19: 615-621, 1983.

Gottardis, M. M., Robinson, S. P., and Jordan, V. C. Estradiol-stimulated growth
of MCF-7 tumors implanted in athymic mice: a model to study the tumoristatic
action of tamoxifen. J Steroid Biochem, 30: 311-314, 1988.

Osborne, C. K., Hobbs, K., and Treat, J. M. Biological differences among MCF-7
human breast cancer cell lines from different laboratories. Breast Cancer Res
Treat, 9: 111-121, 1987.

Gooch, J. L. and Yee, D. Strain-specific differences in formation of apoptotic
DNA ladders in MCF-7 breast cancer cells. Cancer Lett, 144: 31-37, 1999.

Simstein, R., Burow, M., Parker, A., Weldon, C., and Beckman, B. Apoptosis,
chemoresistance, and breast cancer: insights from the MCF-7 cell model system.
Exp Biol Med (Maywood), 228: 995-1003, 2003.

Reddel, R. R., Murphy, L. C., Hall, R. E., and Sutherland, R. L. Differential
sensitivity of human breast cancer cell lines to the growth-inhibitory effects of
tamoxifen. Cancer Res, 45: 1525-1531, 1985.

Amara, J. F., Clackson, T., Rivera, V. M., Guo, T., Keenan, T., Natesan, S.,
Pollock, R., Yang, W., Courage, N. L., Holt, D. A., and Gihnan, M. A versatile
synthetic dimerizer for the regulation of protein-protein interactions. Proc Natl
Acad Sci U S A, 94: 10618-10623, 1997.

Mai, S. Overexpression of c-myc precedes amplification of the gene encoding
dihydrofolate reductase. Gene, 148: 253-260, 1994.

Matsushime, H., Quelle, D. E., Shurtleff, S. A., Shibuya, M., Sherr, C. J ., and

Kato, J. Y. D-type cyclin-dependent kinase activity in mammalian cells. Mol Cell
Biol, 14: 2066-2076, 1994.

75

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

Evan, G. 1., Lewis, G. K., Ramsay, G., and Bishop, J. M. Isolation of monoclonal
antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol, 5:
3610-3616, 1985.

Neff, S., Sadowski, C., and Miksicek, R. J. Mutational analysis of cysteine
residues within the horrnone-binding domain of the human estrogen receptor
identifies mutants that are defective in both DNA-binding and subcellular
distribution. Mol Endocrinol, 8: 1215-1223, 1994.

Shang, Y. and Brown, M. Molecular determinants for the tissue specificity of
SERMs. Science, 295: 2465-2468, 2002.

Wakeling, A. E. Similarities and distinctions in the mode of action of different
classes of antioestrogens. Endocr Relat Cancer, 7: 17-28, 2000.

Pink, J. J. and Jordan, V. C. Models of estrogen receptor regulation by estrogens
and antiestrogens in breast cancer cell lines. Cancer Res, 56: 2321-2330, 1996.

Liu, X. F. and Bagchi, M. K. Recruitment of distinct chromatin-modifying
complexes by tamoxifen-complexed estrogen receptor at natural target gene
promoters in vivo. J Biol Chem, 279: 15050-1505 8, 2004.

Jiang, C., Ito, M., Piening, V., Bruck, K., Roeder, R. G., and Xiao, H. T IP30
interacts with an estrogen receptor alpha-interacting coactivator CIA and
regulates c-myc transcription. J Biol Chem, 279: 27781-27789, 2004.

Keeton, E. K. and Brown, M. Cell Cycle Progression Stimulated by Tamoxifen-
bound ER{alpha} and Promoter-Specific Effects in Breast Cancer Cells Deficient
in the Corepressors N-CoR and SMRT. Mo] Endocrinol, 2005.

Watson, P. H., Pon, R. T., and Shiu, R. P. Inhibition of c-myc expression by
phosphorothioate antisense oligonucleotide identifies a critical role for c-myc in
the growth of human breast cancer. Cancer Res, 51 : 3996-4000, 1991.

Carroll, J. S., Swarbrick, A., Musgrove, E. A., and Sutherland, R. L. Mechanisms
of growth arrest by c-myc antisense oligonucleotides in MCF-7 breast cancer

cells: implications for the antiproliferative effects of antiestrogens. Cancer Res,
62: 3126-3131, 2002.

Swarbrick, A., Akerfeldt, M. C., Lee, C. 8., Sergio, C. M., Caldon, C. E., Hunter,
L. J ., Sutherland, R. L., and Musgrove, E. A. Regulation of cyclin expression and

cell cycle progression in breast epithelial cells by the helix-loop-helix protein Idl.
Oncogene, 24: 381-389, 2005.

Wang, Y. H., Liu, S., Zhang, G., Zhou, C. Q., Zhu, H. X., Zhou, X. B., Quan, L.
P., Bai, J. F ., and Xu, N. Z. Knockdown of c-Myc expression by RNAi inhibits

76

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

MCF-7 breast tumor cells grth in vitro and in vivo. Breast Cancer Res, 7:
R220-228, 2005.

Prall, O. W., Rogan, E. M., Musgrove, E. A., Watts, C. K., and Sutherland, R. L.
c-Myc or cyclin D1 mimics estrogen effects on cyclin E-Cdk2 activation and cell
cycle reentry. Mol Cell Biol, 18: 4499-4508, 1998.

Venditti, M., Iwasiow, B., Orr, F. W., and Shiu, R. P. C-myc gene expression
alone is sufficient to confer resistance to antiestrogen in human breast cancer
cells. Int J Cancer, 99: 35-42, 2002.

Bindels, E. M., Lallemand, F., Balkenende, A., Verwoerd, D., and Michalides, R.
Involvement of G1/ S cyclins in estrogen-independent proliferation of estrogen
receptor-positive breast cancer cells. Oncogene, 21: 8158-8165, 2002.

Cariou, 8., Donovan, J. C., Flanagan, W. M., Milic, A., Bhattacharya, N., and
Slingerland, J. M. Down-regulation of p21WAF 1/CIP1 or p27Kip1 abrogates
antiestrogen-mediated cell cycle arrest in human breast cancer cells. Proc Natl
Acad Sci U S A, 97: 9042-9046, 2000.

Pethe, V. and Shekhar, P. V. Estrogen inducibility of c-Ha-ras transcription in
breast cancer cells. Identification of functional estrogen-responsive transcriptional

regulatory elements in exon l/intron 1 of the c-Ha-ras gene. J Biol Chem, 274:
30969-30978, 1999.

Aktas, H., Cai, H., and Cooper, G. M. Ras links growth factor signaling to the cell
cycle machinery via regulation of cyclin D1 and the Cdk inhibitor p27K1Pl. Mol
Cell Biol, 17: 3850-3857, 1997.

Kasid, A., Lippman, M. E., Papageorge, A. G., Lowy, D. R., and Gelrnann, E. P.
Transfection of v-rasH DNA into MCF-7 human breast cancer cells bypasses
dependence on estrogen for tumorigenicity. Science, 228: 725-728, 1985.

Engel, L. W., Young, N. A., Tralka, T. S., Lippman, M. E., O'Brien, S. J ., and
Joyce, M. J. Establishment and characterization of three new continuous cell lines
derived from human breast carcinomas. Cancer Res, 38: 3352-3364, 1978.

Keydar, 1., Chen, L., Karby, S., Weiss, F. R., Delarea, J ., Radu, M., Chaitcik, S.,
and Brenner, H. J. Establishment and characterization of a cell line of human

breast carcinoma origin. Eur J Cancer, 15: 659-670, 1979.

Engel, L. W. and Young, N. A. Human breast carcinoma cells in continuous
culture: a review. Cancer Res, 38.1 4327-4339, 1978.

Leung, B. S., Qureshi, S., and Leung, J. S. Response to estrogen by the human
mammary carcinoma cell line CAMA-l. Cancer Res, 42: 5060-5066, 1982.

77

55.

56.

57.

Varma, H. and Conrad, S. E. Reversal of an antiestrogen-mediated cell cycle
arrest of MCF-7 cells by viral tumor antigens requires the retinoblastoma protein-
binding domain. Oncogene, 19: 4746-4753, 2000.

Dubik, D. and Shiu, R. P. Mechanism of estrogen activation of c-myc oncogene
expression. Oncogene, 7: 1587-1594, 1992.

Htun, H., Holth, L. T., Walker, D., Davie, J. R., and Hager, G. L. Direct

visualization of the human estrogen receptor alpha reveals a role for ligand in the
nuclear distribution of the receptor. Mol Biol Cell, 10: 471-486, 1999.

78

CHAPTER THREE

c-Myc Suppresses p21WAF U C" 1 Expression During Estrogen Signaling and
Antiestrogen Resistance in Human Breast Cancer Cells

Note: The contents of this chapter have been published in Mukherjee S., and Conrad
SE. 2005. c-Myc suppresses p21WAF 1’ cn>1 expression during estrogen signaling and
antiestrogen resistance in hmnan breast cancer cells. J Biol Chem. Published online
ahead of print, Manuscript: M502278200 (In Press).

79

ABSTRACT
Estrogen rapidly induces expression of the proto-oncogene c-myc. c-Myc is required for
estrogen-stimulated proliferation of breast cancer cells, and deregulated c-Myc
expression has been implicated in antiestrogen resistance. We investigated the
mechanism(s) by which c-Myc mediates estrogen-stimulated proliferation and contributes
to cell cycle progression in the presence of antiestrogen. Using stable MCF-7 derivatives
with inducible c-Myc expression, we demonstrated that in antiestrogen-treated cells the
elevated mRNA and protein levels of p21WAF1/Cm, a cell cycle inhibitor, decreased upon
either c-Myc induction or estrogen treatment. Expression of p21WAF ”cm blocked c-Myc-
mediated cell cycle progression in the presence of antiestrogen, suggesting that the

WAFl/CIPl

decrease in p21 is necessary for this process. Using RNA interference to

suppress c-Myc expression, we further established that c-Myc is required for estrogen-
mediated decreases in p21WAH/CIP'. Finally, we observed that neither c-Myc nor

WAFl/CIPl . . . . .
p21 rs regulated by estrogen or antiestrogen in an antrestrogen-resrstant MCF-7
derivative. The p21WAF”CIPl levels in the antiestrogen-resistant cells increased when c-
Myc expression was suppressed, suggesting that loss of p21WAF1’Cm regulation was a
consequence of constitutive c-Myc expression. Together, these studies identify

IWAFl/ClPl

p2 as an important target of c-Myc in breast cancer cells, and provide a direct

link between estrogen, c-Myc and the cell cycle machinery. They further suggest that
aberrant c-Myc expression, which is frequently observed in human breast cancers, can

1WAFl/CIPl

contribute to antiestrogen resistance by altering p2 regulation.

80

INTRODUCTION

A majority of estrogen receptor (ER)-positive breast tumors require estrogenic
steroids such as l7B-estradiol (E2) for proliferation, and can be treated with antiestrogens
that antagonize the actions of E2 and inhibit tumor growth (1-3). However, many
patients with initially responsive tumors experience recurrence, indicating the
development of acquired antiestrogen resistance (1, 2, 4). The molecular changes
underlying the development of this resistance are poorly understood, and require further
characterization so that new agents that are able to maintain therapeutic effectiveness can
be designed.

E2 mediates passage fi'om G1 to S phase by activating the ER, which functions as
a transcription factor and induces the expression of gene products required for cell cycle
progression (5, 6). Antiestrogens cause a G0/G1 arrest by binding the ER, thus
preventing the activation of genes by E2 (7, 8). Cyclin-dependent kinases (CDKs)
control the Gl/S transition (9-11), and CDK activity is regulated by multiple mechanisms
including phosphorylation, activation by binding of cyclins (cyclin D1-CDK4 or
cyclinE/A-CDKZ), and inhibition by binding of CDK inhibitors such as p21WAFl/Cm
(p21) and p27”' (p27) (12-14). Various proteins involved in CDK regulation have been
identified as potential mediators of E2-induced nritogenesis (15-18), and deregulated
expression of these key targets may enable cells to proliferate in the absence of E2 or the
presence of antiestrogens (18-24).

One important target of E2 in breast cancer cells is the proto-oncogene c-myc (25,
26). The c-Myc protein is a transcriptional regulator, and c-myc antisense

oligonucleotides or c-myc small interfering RNA (siRNA) inhibit E2-stimulated

81

proliferation (27-31). Moreover, ectopic expression of c-Myc is sufficient to induce
proliferation of E2-dependent breast cancer cells in the presence of the antiestrogen ICI
182,780 (ICI) (19, 32). Thus, c-Myc plays a crucial role in mediating E2-regulated
proliferation, and may contribute to antiestrogen resistance.

Among multiple targets of c-Myc identified in diverse cellular systems, several
are key cell cycle regulators (27). Specifically, c-Myc has been reported to increase
expression of positive cell cycle regulators, including cyclin B and CDK4 (33, 34), and to
decrease expression of CDK inhibitors, such as p21 and p27 (35-40). c-Myc could
therefore be mediating its proliferative effects in breast cancer cells by regulating the
levels of some or all of these proteins and their distribution among cyclin/CDK
complexes. However, the target(s) through which c-Myc mediates E2-stimulated
proliferation of breast cancer cells, and can contribute to proliferation in the presence of
antiestrogen, have not been identified. In this study, we sought to identify these target(s)
using the MCF-7 cell line, a model of E2-dependent and antiestrogen-sensitive human
breast cancer (41, 42), and stably transfected MCF-7 derivatives in which induction of
ectopic c-Myc could promote cell cycle progression in the presence of ICI.

We examined several cell cycle proteins reported to be targets of c-Myc, and
established that c-Myc induction in ICI-treated cells decreased expression of the CDK
inhibitor p21 to levels seen in E2-treated cells. We further showed that this decrease was
a consequence of downregulated p21 mRNA levels, and that c-Myc could repress p21
promoter activity. Expression of p21 from an adenoviral vector blocked c-Myc-mediated
cell cycle progression of ICI-treated cells, suggesting that the decrease in p21 was

important for this process. It has been reported that E2 treatment of MCF-7 cells leads to

82

decreased p21 expression (18, 22). Using RNA interference to knock down c-Myc
expression, we have determined that c-Myc is required for the E2-mediated decrease in
p21, thus providing a direct link between E2, c-Myc and the cell cycle machinery. A
previous study demonstrated that decreasing p21 levels with antisense oligonucleotides is
sufficient to cause cell cycle progression of ICI-treated cells (22). Based on this finding
and our current results, we propose that a key mechanism by which c—Myc promotes
proliferation of breast cancer cells in the presence of ICI is by decreasing p21 levels.

To determine whether deregulation of c-Myc is associated with acquired
antiestrogen resistance, we examined LCC9, an MCF-7 derivative selected for both E2
independence and ICI resistance (43). We observed that neither c-Myc nor p21
expression was affected by E2 or ICI treatment of LCC9 cells. However, p21 levels in
LCC9 cells increased when c-Myc expression was suppressed using RNA interference.
This indicated that the loss of p21 regulation in LCC9 cells is likely to be a consequence
of constitutive c-Myc expression. These results provide fiuther evidence that aberrant
regulation of c-Myc and/or p21 could play a role in the progression of tumors to an

antiestrogen-resistant phenotype.

MATERIALS AND METHODS
Cell culture. MCF-7 and LCC9 cells were obtained from the Lombardi Cancer Center.

Cells were maintained in improved modified Eagle’s medium (IMEM) (BioFluids, Inc.)

supplemented with 5% fetal bovine serum (FBS) (HyClone), 100 units/ml penicillin

83

(Invitrogen), and 100 units/ml streptomycin (Invitrogen), and cultured at 37°C with 5%
C02. MCF-7/Myc33 and MCF-7/Myc31, stably transfected MCF-7 derivatives, were
constructed as described in Chapter two, and were maintained in IMEM with 5% FBS,
penicillin/streptomycin, Geneticin (Sigma) (50 rig/m1), and Hygromycin (Invitrogen) (10
ug/ml). When studying the effects of E2 and ICI, cells were cultured in E2-free medium,
which is phenol red-free IMEM (BioFluids, Inc.) containing 5% charcoal-stripped fetal
bovine serum (CSS) (HyClone), and penicillin/streptomycin. For most experiments, cells
were plated in medium with FBS for 24 h, and pre-arrested for 48 h in CSS containing
medium with 10 nM ICI (Zeneca Pharmaceuticals). Cells were then treated with CSS
containing medium with either 10 nM E2 (Sigma), 10 nM ICI + 300 nM AP1510 (AP), to
induce c-Myc, or 10 nM ICI alone, and harvested at various time points. For some
experiments, cells were not pre-arrested with ICI in order to improve transfection
efficiency or to facilitate comparisons between MCF-7 cells and ICI-resistant LCC9 cells.
In these experiments, cycling cells were directly treated with medium containing either
100 nM ICI or 1 nM E2, and then harvested. The higher ICI concentration was used to
observe rapid arrest in cycling cells, and 1 nM E2 was used since cells were not being

released from an ICI arrest.

Plasmids. The p15 plasrrrid, containing human c-myc cDNA (exons 2 and 3), was a gift
from Dr. S. Mai (44). The c-myc fragment was excised fi'om pJ 5 using HindIII and Bng,
and used as a probe for Northern blot hybridizations. The p21 cDNA probe used for
Northern blotting was excised fi'om the pCEP-WAF 1-S plasmid, and was obtained fi'om

Dr. L.K. Olson (45). The pSP271 vector and the pSP271-Myc expression plasmid were a

84

gift from Dr. R.N. Eisenman (46). The -l94 p21 promoter-luciferase plasmid was

obtained from Dr. H.R. Kim with permission from Dr. X.F. Wang (47).

Western Blotting. Cells were lysed as described previously (48), and protein in the cell
lysates was quantitated using the Bradford protein assay (Bio-Rad). Proteins (10-50 ug)
were resolved by SDS-PAGE (7.5% for retinoblastoma protein (pRB)
hyperphosphorylation and 12% for all other proteins analyzed), transferred to
polyvinylidene difluoride membranes (Perkin Elmer), and probed with primary
antibodies for c-Myc (clone 9E10, ATCC) (49), cyclin A (clone BF683, Pharrrringen),
pRB (clone G3-245, Pharrningen), cyclin D1 (UBIO6137, Upstate Biotechnology, Inc.,),
cyclin E (clone HE12, Santa Cruz Biotechnology), p21 (p21-C-19, Santa Cruz
Biotechnology), p27 (p27-C-19, Santa Cruz Biotechnology), CDK4 (clone H22, Santa
Cruz Biotechnology), or B-actin (clone AC-40, Sigma). After washing, membranes were
incubated with horseradish peroxidase-conjugated goat anti-rabbit (Bio-Rad) or goat anti-
mouse (American Qualex) secondary antibodies. Bands were visualized using Super

Signal West Pico Chemiluminescent Substrate (Pierce).

Infections with Recombinant Adenoviruses. A recombinant adenovirus (AdCMV)
containing the cDNA encoding human p21 (Ad-p21) and a control virus encoding [3-
galactosidase (Ad-B-gal) were obtained from Dr. L.K. Olson (45). Cells were plated at
106 cells/IOO-mm dish in medium with FBS for 24 h, and then treated with css
containing medium with 10 nM ICI. After 24 h, this medium was removed and saved.

The Ad-p21 or Ad-B-gal viruses were diluted in CSS containing medium, and cells were

85

infected with 5 plaque-forming units (pfu) per cell in a total volume of 1.0 ml. Infections
were carried out at 37°C for 2 h. Following infection, the medium was replaced with the
original medium containing 10 nM ICI. The cells were pre-arrested for another 24 h, and
then treated with 10 nM ICI + 300 nM AP. After 24 h the cells were harvested and

subjected to cell cycle analysis and Western blotting.

Northern Blotting. Cells were lysed, and total RNA was purified using Trizol reagent
(Invitrogen). Ten micrograms of RNA were electrophoresed on 1% formaldehyde-
agarose gels, transferred to nitrocellulose membranes (Schleicher and Schuell), and UV-
cross-linked. The blots were then hybridized with a 32P-labeled cDNA probe for c-myc
or p21. The membranes were stripped and reprobed for GAPDH as a loading control.
Phosphorlmager scanning (Amersharn Biosciences) was used to quantitate the bands

obtained, and the c-myc or p21 mRNA levels in each sample were normalized to

GAPDH.

Luciferase Assays. MCF-7 cells were plated at 5 x 105 cells/60-mm dish. After 24 h
cells were transfected using Lipofectin reagent (Invitrogen). One ug of pSP27l-Myc, or
pSP271 vector was co-transfected with 0.25 pg of either -194 p21 promoter-luciferase
plasmid or pGL2Basic vector. Al] transfections also included 0.1ug of pCMV-B-
galactosidase which served as a control for transfection efficiency. Transfections were
carried out for 5 h in serum free medium (SFM), followed by incubation overnight in
complete medium containing FBS. Cells were then treated with CSS containing medium

with 100 nM ICI and harvested after 24 h. Both luciferase and B-galactosidase activities

86

were measured using the protocol provided by the manufacturer (Promega, Clontech) on
a Turner TD 20E luminometer (Turner Designs). Each transfection was done in
triplicate, and the luciferase activity was normalized to B-galactosidase activity in each

sample.

Cell Cycle Analysis. Cells were trypsinized, washed in phosphate buffered saline (PBS),
suspended in PBS + 10% FBS, fixed with 80% cold ethanol and stored at -20°C. Prior to
analysis, cells were washed twice with PBS, and suspended in PBS containing 1 mg/ml
RNAase A, 0.2 mg/ml propidium iodide, 0.5 mM EDTA and 0.1% Triton X-100. Cells
were then analyzed for red fluorescence on a FACSVantage flow cytometer, and cell

cycle was distribution determined using ModFitLT software.

Gene Silencing with small interfering RNAs (siRNAs). MCF-7 or LCC9 cells were
cultured in 6-well plates containing 3 ml of medium with FBS. After 24 h, cells were
transfected with 100 nM of c-myc or negative control siRNA duplexes (Ambion), using
siPORT TM Lipid Transfection Agent (Ambion), according to manufacturer’s
recommendations. Transfections were carried out for 4 h in SFM, followed by treatment
with CSS containing medium with either 1 nM E2 or 100 nM ICI. Cells were harvested
after 48 h, and the cell lysates analyzed by Western blotting. Cells maintained in SFM
for 4 h and harvested after treatment with 100 nM ICI or 1 nM E2 for 48 h served as

untransfected controls.

87

RESULTS

p21 is targeted during Myc-induced cell cycle progression of MCF-7 cells in the
presence of ICI. Time course experiments were carried out to investigate whether, upon
c-Myc induction, changes occur in the levels of key cell cycle regulators that might
contribute to cell cycle progression. MCF-7/Myc33 cells were pre-arrested with ICI,
treated with ICI, ICI+AP or E2, and harvested at 6 h intervals for analysis by flow
cytometry and Western blotting. The distribution of cells in different phases of the cell
cycle was determined, and the data for S phase is shown in Fig. 1A. Fewer than 5% of
ICI-treated cells were in S phase, and no significant changes were seen throughout the
time course. Cell cycle phase distributions and kinetics of cell cycle progression were
similar for ICI+AP- and E2-treated Cells over the duration of this experiment.

As shown in Fig. 1B, ICI+AP treatment of pre-arrested MCF-7/Myc33 cells
caused an increase in c-Myc protein within 6 h to levels similar to those seen in E2-
treated cells, and its levels remained elevated throughout the time course. Thus, c-Myc
expression at levels comparable to those seen in E2 is sufficient for cell cycle progression
in the presence of ICI. Since cyclin A expression increases at the Gl-S transition, it
serves as a marker for cell cycle progression (50). In these experiments, there was a good
correlation between cyclin A protein expression and S phase entry at 18 h.

To identify proteins that are regulated by c-Myc, the levels of key cell cycle
regulatory proteins were compared in the three treatments (Fig. 1B). Cyclin D1 remained
high in E2-treated cells, and decreased upon ICI treatment. This decrease was not

prevented by c-Myc induction in ICI+AP-treated cells, which is consistent with a

88

Figure 1: Effects of c-Myc induction on cell cycle progression and protein
expression in ICI-treated cells, and in cells infected with an adenovirus expressing
p21. MCF-7/Myc33 cells were pre-arrested with ICI, then treated with ICI, ICI+AP or
E2, and harvested at 6 h intervals. A) Cell cycle analysis was performed as described in
“Materials and Methods”. The average percentage of cells in S phase for each time point
is shown +/- standard deviation. A, E2-treated cells. I, ICI+AP-treated cells. 0, ICI-
treated cells. B) Cell lysates were analyzed by Western blotting for c-Myc, cyclin A,
cyclin D1, cyclin B, CDK4, p21, p27, and actin. These experiments were repeated once
with similar results. C) The experiments described in A) and B) were also carried out
with the MCF-7/Myc 31 cell line. Western blot results for p21 and actin are shown. D)
Control cells stably transfected with the vector were pre-arrested with ICI, and then
treated with ICI, ICI+AP or E2 for 24 h. Cells were harvested and the levels of c-Myc
(data not shown), p21 and actin were determined by Western blotting. E) MCF-7/Myc 33
cells were pre-arrested with ICI for 24 h, and then infected with adenoviruses encoding
either p21 (Ad-p21) or B-galactosidase (Ad-B-gal) at 5 pfu/cell for 2 h. Arrest with ICI
was continued for another 24 h, followed by treatment with ICI+AP. After 24 h, cells
were harvested and cell cycle analysis was performed. The average percentage of cells in
S phase is shown +/- standard deviation. F) The cells infected with Ad-p21 or Ad-B-gal
as described in E) were harvested concurrently with those used for cell cycle analysis,
and the levels of p21 and actin were determined by Western blotting.

89

 

 

 

 

 

 

A.
g 35
g 30*
(D 25.
.5
=9. 20.

8 15.
3 10-

C

8 5-

a“: o
n- I I l I l I

0 6 12 18 24 30 36
Duration of treatment (h)
B.
ICI+AP 7 E2 lCl _

0 612182430612182430612182430 'I'Ime(h)
it??? ‘3" “‘~ :‘2. .2: 2.2::- 1; 4‘3 c-Myc
if 1"" .1.-. '- 7.5-2 -_ .1 cyclinA

1.... fir-IT ”.7 .-7-7.____,_,,_._,._ __,_, _: cyclinD1

~ “2 "L": .fim: '12-..- ‘3

new ”'51:.

.14", h“ Adler-mum “92%: p21
{23 ¥WZ$M$ :1. fl p27

“m b- — - - ——— - Actin
,- wuss-vim: ntam .- vase. 4‘...- 1,937 3;, wm:gw

 

 

ICI+AP E2 ICI
0 6121824303661218 243036 61218 243036 Time(h)
22.-22:22::17:.5“: "" ‘.*'.: :1; 4:1 p21
«pH—tn-..“ .wmw.-.........- a...“ m _u w.— - .. a.
‘W-M ACIII'I
E.
8
2 18 .4...

 

 

 

 

Ad-p21 Ad-B—gal

90

Vector Control

_I_C| E_2
AP

F“: :1 p21

I:- - ‘2 Actin

Ad- Ad-
p21 B-gal
'1' p21

"’ ‘1'1Actin

previous report that c-Myc does not induce proliferation by increasing cyclin D1 (19).
Although cyclin B and CDK4 are constitutively expressed in MCF-7 cells, their levels
were examined since they have been implicated as targets of c-Myc in other systems (33,
34). Ectopic c-Myc expression in the presence of ICI did not alter the levels of cyclin E
or CDK4.

Next, the CDK inhibitors p27 and p21 were examined. In this experiment, p27
levels were decreased at 6-12 h in E2-treated cells as compared to ICI-treated cells. The
expression pattern of p27 in ICI+AP-treated cells was similar to that in ICI-treated cells,
and though subtle decreases in p27 levels occurred at 24-30 h in ICI+AP-treated cells,
these decreases were not consistently observed over several experiments. Therefore, p27
is unlikely to be a major cause of c-Myc-mediated S phase entry. Expression of p21 was
high in ICI-treated cells, and c-Myc induction caused a dramatic decrease to levels
comparable to those seen in E2-treated cells. This decrease was consistently apparent by
12-18 h in several independent experiments, and preceded major changes in the
percentage of cells in S phase.

To establish that this result was not unique to MCF-7/Myc33 cells, a second
independently derived transfectant, MCF-7/Myc3l, was examined. As shown in Fig. 1C,
the decrease in p21 levels in MCF-7/Myc 31 cells was similar to that seen in the MCF-
7/Myc33 cell line, and occurred by 18 h of treatment with either E2 or ICI+AP. Cell
cycle progression, and both c-Myc and cyclin A protein levels were also examined in
MCF-7/Myc31 cells, and the results were similar to those obtained with MCF-7/Myc33
cells (data not shown). In control cells stably transfected with vector alone, p21 levels

decreased by 24 h of E2 treatment, but remained high after ICI+AP treatment (Fig. 1D),

91

establishing that the decrease in p21 was specific to c-Myc induction in the presence of
ICI.

The data presented established that expression of c-Myc in the presence of ICI
leads to significant changes in the levels of two proteins, p21 and cyclin A. Decreasing
p21 expression using antisense oligonucleotides is sufficient to induce cell cycle
progression in ICI-treated cells (22), while induction of cyclin A alone using the AP
inducible system is not (Zhang C., Conrad S.E., unpublished data). We therefore chose
to focus on p21 in ftuther experiments by determining whether this decrease in p21 is
necessary for c-Myc-mediated cell cycle progression in the presence of antiestrogens, and

by characterizing the mechanism by which c-Myc downregulates p21 expression.

p21 expression can block c-Myc-mediated cell cycle progression in the presence of
ICI. To determine whether a decrease in p21 levels is required in order for c-Myc to
promote cell cycle progression in the presence of ICI, pre-arrested MCF-7/Myc33 cells
were infected with adenoviruses encoding either p21 (Ad-p21) or B-galactosidase (Ad-[3-
gal). The Ad-B-gal virus was used as a control for possible effects of viral infection.
Cellswere then treated with ICI+AP to induce c-Myc expression. Western blot analyses
confirmed that p21 was expressed in Ad-p2l-infected but not in Ad-B-gal-infected cells
(Fig. 1F). The percentage of cells in S phase was ~2.5 fold lower in cells infected with
Ad-p21 than Ad-B-gal (Fig. 1B). This indicated that p21 expression blocks the ability of
c-Myc to promote cell cycle progression in the presence of antiestrogen, and suggested
that the c-Myc-mediated decrease in p21 was necessary for this process. Our results

complement previous studies which have demonstrated that p21 is a critical regulator of

92

CDK activity in human breast cancer cells, and that promoting the formation of p21 free
CDK complexes is sufficient for CDK activation and cell cycle progression (16, 17, 51,
52). Together, these results suggests that a key mechanism by which c-Myc promotes

proliferation of breast cancer cells in the presence of ICI is by decreasing p21 levels.

c-Myc expression leads to decreased p21 mRNA levels and promoter activity in
MCF-7 cells. To characterize the mechanism(s) by which c-Myc and E2 downregulate
p21 in MCF-7 cells, p21 mRNA and protein were examined in tandem. MCF-7/Myc33
cells were pre-arrested with ICI and then treated with ICI, ICI+AP or E2. At various
times after treatment, cells were harvested and the levels of p21 mRNA and protein were
analyzed by Northern and Western blotting, respectively. Relative to the ICI-treated
cells, a decrease in p21 mRNA levels was seen at 12 h in both ICI+AP- and E2-treated
cells, and a decrease in p21 protein levels was apparent by 18 h (Figs. 2A-B). Using
phosphorimager analysis, the decrease in p21 mRNA at 24 h was determined to be 2-3
fold relative to the 12 h ICI treated sample (Fig. 2A, also see Fig. 5C).

To accurately quantitate the decrease in p21 protein levels, the 24 h ICI-treated
sample was diluted as indicated and the intensities of the p21 bands were compared to
those of the undiluted 24 h ICI+AP- and E2-treated samples by Western blotting (Fig.
2C). Actin protein levels served as controls and demonstrated the accuracy of the
dilutions. The p21 levels in the ICI+AP- and E2-treated samples were lower than the 2-
fold but higher than the 4-fold diluted ICI samples. Thus, the decrease in p21 protein
levels caused by ectopic c-Myc expression or E2 treatment is approximately 3-fold, and

is similar to the reduction in mRNA. These results concur with a prior report which

93

Figure 2: Analysis of p21 protein and mRNA levels, and p21 promoter-luciferase
activity following c-Myc induction in ICI-treated cells. MCF-7/Myc33 cells were pre-
arrested with ICI, then treated with ICI, ICI+AP or E2 and harvested at the indicated
times. A) RNA was isolated and Northern blotting was carried out as described in
“Materials and Methods”. Bands were quantitated by phosphorimaging. The p21 mRNA
levels were normalized to the GAPDH levels in each sample, and are represented as
expression relative to the 12 h ICI treatment. B) Cell lysates were prepared and analyzed
for p21 and actin protein levels by Western blotting. C) The 24 h ICI sample was diluted
as indicated, and the intensity of the p21 bands were compared to those seen in the
undiluted 24 h ICI+AP and E2 samples. This experiment was repeated once with similar
results. In both experiments, expression of c-myc mRNA and protein was confirmed
(data not shown). D) MCF-7 cells were co-transfected with a pSP27l-Myc expression
plasmid or pSP271 vector, and either -l94 p21 promoter-luciferase plasmid or
pGL2Basic vector. All transfections also included a CMV promoter-driven B-
galactosidase gene as a control for transfection efficiency, and luciferase activity was
normalized to B-galactosidase activity in each sample. The results shown represent fold
change compared to the pSP27l vector control, and are the average +/- standard deviation
of three independent experiments, each done in triplicate.

94

 

 

12 18 24 Time (h)
lCl 101 E2 ICI ICI E2 ICI ICI £2
+AP +AP +AP

Qfléiéphnh p21

’1
‘IOOUIUIQQ GAPDH

1 0.7 0.7 0.8 0.60.5 0.9 0.50.4 Relative
Expression

12 18 24 Time (h)
ICI 101 E2 lCl lCl E2 ICI ICI E2
7 .+.AP +AP +AP
J ‘ 5,: .021

$“- ',~" '92—0 Actin

0' ICI ICI E2

+AP
1 101/4"? 1 1 Dilution
‘1'; TAu’T"‘ v“ ail-v3.71 m4:
:E't.’...73_.3. $.34. p21

 

D.
a .
221.001 :33
3 , 2.3-4

0.80 .
i : {:3
*3 0.60: 11.2!"
:r l w:
_l .
.E l i
a 0.40I ,1
C . I,
2.

‘ ‘ ‘ _ 0.01

E0000 22.33%. _E_ 2..

c-Myc Vector c—Myc Vector

-194 p21 promoter Luciferase Rector
luciferase

95

demonstrated that E2 decreases p21 mRNA levels (53). In addition, previous studies in
other systems indicate that c-Myc can repress transcription from the p21 promoter (35-
39), and our Northern blot results suggest that similar mechanisms may be operative in
MCF-7 cells.

To directly test whether c-Myc can repress p21 promoter activity in MCF-7 cells,
we co-transfected cells with a c-Myc expression vector and a p21 promoter-luciferase
reporter construct. Previous studies have shown that the p21 promoter region
downstream of -119 bp is important for regulation by c-Myc (37), and footprinting
experiments indicated that c-Myc binds to this region of the promoter (35). We therefore
used a p21 promoter fragment beginning at -194 bp in our experiment. Expression from
this promoter was decreased more than two-fold in cells co-transfected with the c-Myc
expression vector (Fig. 2D). The control promoter-less luciferase plasmid showed very
low levels of activity and these levels were not regulated upon co-transfection with c-
Myc. The level of repression observed in these luciferase assays is similar to that seen in
other systems (35, 36, 38, 39), and is consistent with both our Northern and Western blot

analyses.

E2 mediates downregulation of p21 through c-Myc. Both c-Myc and p21 are
established targets of E2 in breast cancer cells (22, 25, 26), and the results described
above demonstrate that c-Myc expression is sufficient to decrease p21 levels in the
presence of ICI. Together, these facts suggest that the decrease in p21 caused by E2 is

mediated primarily through c-Myc. To test this hypothesis, we suppressed c-Myc

96

ICI E2 E2 E2
+msi +csi

i - a- - c-Myc
_fld'i

L”: ‘m- n'...le_

 

 

Jul _' tun-e. 'g-Fb'.!:hf «as!
1.. - ---. - .. fat; [32‘]

L. 44-
F .a't‘rt ' M -9-

.——-: — n ACIIII

Figure 3: Effect of c-myc siRNA on p21 expression in E2-treated MCF-7 cells.
Cycling MCF-7 cells were transfected with 100 nM c-myc siRNA (msi) or control siRNA
(csi) duplexes as described in “Materials and Methods”, and then treated with E2. Cells
were harvested after 48 h and proteins were analyzed by Western blotting. Levels of c-
Myc, p21 and actin were compared to those seen in untransfected cells treated with ICI or
E2. This experiment was repeated twice with similar results.

97

expression using RNA interference and assayed whether E2 can decrease p21 in the
absence of c-Myc.

MCF-7 cells were transfected with either c-myc or control siRNA, and then
incubated in the presence of E2. After 48 h, cells were harvested and the levels of c-Myc
and p21 were determined by Western blotting. As shown in Fig. 3, c-myc siRNA but not
control siRNA decreased c-Myc protein in E2-treated cells to levels similar to those in
ICI-treated cells. As a result, the levels of p21 in cells transfected with c-myc siRNA
were similar to those in ICI-treated cells. This demonstrates that c-myc and p21 are not
independent targets of E2, but rather that the decreased p21 expression caused by E2 is

mediated primarily through c-Myc.

c-Myc expression is deregulated in the antiestrogen-resistant LCC9 cell line. As
shown by the above results (Fig. l) and others (19, 32), aberrant c-Myc expression
promotes proliferation of MCF-7 cells in the presence of ICI, suggesting that it could
contribute to antiestrogen resistance. To determine whether the regulation of c-Myc
expression is altered in cells with acquired antiestrogen resistance, we examined the
LCC9 cell line, an ICI-resistant MCF-7 derivative (43).

MCF-7 and LCC9 cells were pre-treated with ICI for 48 h, and then incubated
with medium containing either ICI or E2 and harvested every 24 h for 72 h. As shown in
Fig. 4A, c-Myc levels were low in ICI-treated MCF-7 cells and were induced by E2
treatment. In contrast, c-Myc was expressed at sirrrilar levels in both E2- and ICI-treated
LCC9 cells. pRB is a substrate for G1 CDKs, and hyperphosphorylation of pRB

precedes S phase entry (54). Hence, both cyclin A and hyperphosphorylated pRB serve

98

A. MCF-7 LCC9
' ICI E2 " ICI E2 7
o 24 48 72 24 48 72 0 24148 72 24 48 72 Tme (h)
H" 'V‘ ;‘ lI:
weh-—-——rfl c-Myc

partlf‘
‘au4¢‘u,x

 

M_§__F-7 L___CC9
ICI E2 ICI E2

13337131 °""V°

.0

 

romance

_6

A . 0
01 O 0" O 01 001

Fold change in c-myc mRNA levels

 

 

0.0

 

 

MCF-7 LCCQ

Figure 4: Analysis of c-Myc expression in an antiestrogen-resistant cell line. A)
Cycling MCF-7 and LCC9 cells were pre-treated with ICI for 48 h, then treated with
either ICI or E2 and harvested every 24 h for 72 h. c-Myc, cyclin A, pRB and actin
protein levels were analyzed by Western blotting. ppRB denotes the
hyperphosphorylated form of pRB, which migrates more slowly than the
hypophosphorylated form marked pRB. B) Cycling MCF-7 and LCC9 cells were treated
with either ICI or E2 for 48 h and harvested. RNA was prepared and analyzed for c-myc
and GAPDH mRNA levels by Northern blotting. C) Bands seen in (B) were quantitated
by phosphorimaging. c-myc mRNA levels were normalized to GAPDH levels in the
same sample, and are represented as fold increase over the ICI-treated MCF-7 sample.
The graph represents the results as the mean +/- standard deviation of three independent
experiments.

99

as markers for CDK activity and cell cycle progression. Deregulated c-Myc expression
in LCC9 cells correlated with their ability to proliferate in the presence of ICI, as
indicated by both pRB hyperphosphorylation and cyclin A expression.

To determine if the deregulated c-Myc protein expression in LCC9 cells was a
result of altered mRNA levels, MCF-7 and LCC9 cells were treated with ICI or E2 for 48
h, and c-myc mRNA levels were analyzed by Northern blotting. Expression of c-myc
mRNA was high in E2 and low in ICI-treated MCF-7 cells, and this regulation was lost in
LCC9 cells (Fig. 4B). Data from three independent experiments indicated that the levels
of c-myc mRNA in ICI-treated MCF-7 cells were at least 2-fold lower than in all other
samples (Fig. 4C). Together, these results established that c-Myc mRNA- and protein are
expressed at similar levels in E2- and ICI-treated LCC9 cells, and that these levels are
similar to those in E2-treated MCF-7 cells. This suggests that deregulation of c-Myc
expression may contribute to or causes the acquired antiestrogen-resistant phenotype of

LCC9 cells.

ICI does not increase p21 mRNA levels in LCC9 cells. The results shown in Figs. 1-3
indicated that c-Myc decreases both p21 mRNA and protein levels in MCF-7 cells, and
that E2 treatment causes a decrease in p21 through c-Myc. We also showed that c-Myc
expression is deregulated in LCC9 cells (Fig. 4). Previous experiments in our laboratory
had indicated that CDK activity in LCC9 cells was resistant to ICI treatment, and that this
resistance was correlated with aberrant regulation of p21 protein levels (Skildum A., et

81., unpublished data). To determine whether altered regulation of p21 mRNA expression

100

A.
MCF-7 LCC9
_0_ 48 _0 48 Time (h)
FBS ICI E2 FBS ICI E2
pd*'-. 21;“ __‘T 771 p21

 

-.__Y',,_._,F-;_

MCF-7 L009
'_0_ 48 '_o_4 48 Time(h)
FBS ICI E2 FBS ICI “E2_
Féf 5“ a 1”: Q ”R" 3: p21

uflfi56'52;- ' J A

—..-_.q_

11, .2‘ _p: Q B .t ‘ GAPDH

 

 

 

Fold change in p21 mRNA levels

 

 

Figure 5: Analysis of p21 protein and mRNA levels in LCC9 cells. Cycling MCF-7
and LCC9 cells in FBS-containing medium were harvested at 0 h before treatment, or
treated with ICI or E2 for 48 h and then harvested. A) p21 and actin protein levels were
analyzed by Western blotting. B) p21 and GAPDH mRNA levels were determined by
Northern blotting. C) Bands seen in (B) were quantitated by phosphorimaging. p21
mRNA levels were normalized to GAPDH levels in the same sample, and are represented
as fold increase over the E2-treated MCF-7 sample. The graph represents the results as
the mean +/- standard deviation of three independent experiments.

101

accounted for the changes in p21 protein levels in LCC9 cells, we conducted tandem
Northern and Western blot analyses.

Cycling MCF-7 and LCC9 cells in FBS-containing medium were harvested
before treatment (0 h), or treated with ICI or E2 for 48 h and then harvested. The levels
of p21 protein and mRNA were examined by Western and Northern blotting, respectively
(Figs. 5 A—B). In MCF-7 cells, p21 protein and mRNA levels were low at O h, since the
FBS-containing medium has sufficient E2 to support proliferation. The levels of p21
were increased by 48 h of ICI treatment, but remained low in the E2 treatment. In
contrast, p21 protein and mRNA were expressed at similar levels in both E2- and ICI-
treated LCC9 cells. The p21 mRNA levels were approximately 3-fold higher in ICI-
treated MCF-7 cells than in all other samples (Fig. 5C), demonstrating that p21 mRNA
expression increases in response to ICI treatment in MCF-7 cells, but not in LCC9 cells.
These results indicate that the aberrant p21 protein levels observed in LCC9 cells are
likely a result of altered regulation of p21 mRNA, which in turn may be the result of

deregulated c-Myc expression.

p21 levels in LCC9 cells are regulated by c-Myc. The results in Figs. 4-5 demonstrated
that neither c-Myc nor p21 levels are regulated by E2 or ICI in LCC9 cells, and the data
in Figs. 1-3 showed that p21 is a target of c-Myc in MCF-7 cells. This suggested that the
loss of p21 regulation in LCC9 cells is a consequence of constitutive c-Myc expression.

To test this hypothesis, RNA interference was used to knock down c-Myc expression in

LCC9 cells.

102

LCC9 MCF-7

lCl ICI E2 ICI E2 E2 E2
+msi +msi +csi

 

'9. til-V

“ '. q
fit-564- sigma-Myc
.- . --- p21

b---“.’ Actin

- n. I

Figure 6: Effect of c-myc siRNA on p21 expression in LCC9 cells. Cycling LCC9 or
MCF-7 cells were transfected with 100 nM c-myc siRNA (msi) or control siRNA (csi)
duplexes as described in “Materials and Methods”, and then treated with E2 or ICI. Cells
were harvested after 48 h and proteins were analyzed by Western blotting. Levels of c-
Myc, p21 and actin were compared to those seen in untransfected cells treated with ICI or
E2. This experiment was repeated once with similar results.

103

LCC9 were transfected with c-myc siRNA and then incubated in the presence of
ICI. MCF-7 cells served as controls, and were transfected with either c—myc or control
siRNA and incubated in the presence of E2. After 48 h, cells were harvested and the
levels of c—Myc and p21 were determined by Western blotting (Fig. 6). As expected, c-
Myc expression was not decreased in ICI-treated relative to E2-treated LCC9 cells, and
the levels were equivalent to those seen in E2-treated MCF-7 cells. Treatment with c-
myc siRNA decreased c-Myc expression in both ICI-treated LCC9 cells and E2-treated
MCF-7 cells, to levels similar to ICI-treated MCF-7 cells. This decrease was specific to
c-myc siRNA, as it was not seen when cells were treated with control siRNA. In
agreement with the results shown in Fig. 5B, p21 levels did not increase in ICI-treated
relative to E2-treated LCC9 cells, and were comparable to those in E2-treated MCF-7
cells. However, in LCC9 cells transfected with c-myc siRNA in the presence of ICI, p21
expression increased and was similar to that seen in ICI-treated MCF-7 cells. This
demonstrates that p21 can be regulated by c-Myc in LCC9 cells, and suggests that the
loss of p21 regulation by E2 and ICI in these cells is a consequence of constitutive c-Myc

expression.

DISCUSSION

Various mechanisms have been proposed to account for acquired antiestrogen
resistance in breast cancer cells, but since E2 and antiestrogens control cell cycle

progression (55, 56), the development of resistance must ultimately be associated with

104

changes in cell cycle regulation. Numerous studies support the idea that antiestrogen
resistance can result from deregulated expression and/or activity of cell cycle regulatory
proteins, including positive regulators such as cyclin D1 and cyclin B, and negative
regulators such as p21, p27 and pRB (19-24). c-Myc is rapidly induced upon E2
treatment of responsive cells, and it can impinge upon the cell cycle machinery (25-27).
c-Myc is required for E2-stimulated proliferation, and its expression alone is sufficient to
induce cell cycle progression in the presence of antiestrogens (19, 28, 29, 32). Although
c-Myc has been extensively studied in many systems, the targets through which it
mediates E2-stimulated proliferation and contributes to cell cycle progression in the
presence of antiestrogens in breast cancer cells have not been identified.

We established stable MCF-7 derivatives in which ectopic c-Myc expression
could be tightly regulated and could induce cell cycle progression in the presence of ICI.
As discussed previously, we see variability with regard to responsiveness of cells to E2,
possibly due to variation in the amount of other mitogens present in the batches of CSS
used for treatment. In the experiment shown in Figure 1A, the percent of E2-treated
MCF-7/Myc cells in S phase at 24 h was 27% as against 50% observed previously
(Figure 5D and 6A, Chapter 2). In contrast, treatment of cells with ICI+AP, reproducibly
resulted in approximately 20% of cells entering S phase at 24 h.

To identify the target(s) through which c-Myc mediates cell cycle progression in
the presence of ICI, we induced c-Myc expression in ICI-treated cells and examined the
expression of key cell cycle regulators reported to be targets of c-Myc in other cell types
(27). Among the proteins tested, c-Myc caused dramatic changes in the levels of two

proteins, p21 and cyclin A (Fig. 1). Decreasing p21 expression using antisense

105

oligonucleotides is sufficient to induce cell cycle progression in ICI-treated cells (22),
while induction of cyclin A using the AP inducible system is not (Zhang C., Conrad S.E.,
unpublished data). In addition, previous work has demonstrated that p21 is a critical
regulator of CDK activity in human breast cancer cells, and that promoting the formation
of p21 fiee CDK complexes is sufficient for CDK activation and cell cycle progression
(16, 17, 51, 52). This suggests that a key mechanism by which c-Myc promotes
proliferation of breast cancer cells in the presence of ICI is by decreasing p21 levels.
Further support for this viewpoint is provided by the fact that expression of p21 from an
adenoviral vector suppresses the ability of c-Myc to promote cell cycle progression in the
presence of ICI (Fig. 1E, F).

Our results with stable MCF-7 derivatives indicated that c-Myc can repress p21
expression, but did not directly address whether the previously reported (18, 22, 51)
decreases in p21 in response to E2 treatment are mediated via c-Myc. We therefore used
RNA interference to demonstrate that c-Myc expression is required for the E2-mediated
decrease in p21 in MCF-7 cells, and thus identified c-Myc as the first direct link between
E2 and p21 in breast cancer cells. Together, our studies suggest a model (Fig. 7) for
antiestrogen-sensitive cells, in which E2 induces c-Myc, which in turn represses p21 and
results in CDK activation and cell cycle progression. Antiestrogens such as ICI repress
c-Myc expression in these cells, thereby leading to increased levels of p21, CDK
inhibition, and cell cycle arrest. Antiestrogen resistance could arise if c-Myc expression
is no longer inhibited by antiestrogens, as this would lead to decreased p21 levels, CDK

activation, and cell cycle progression.

106

A.

E2 ——> c-Myc—-| p21 —> CDK —>S phase
activation entry

B.

ICI _.._4 c-Myc—> p21—l CDK —-l s phase

. . t
Antiestrogen activation en ry

resistance

Figure 7: Proposed model for the role of c-Myc in E2 signaling and antiestrogen
resistance. A) In antiestrogen sensitive cells, E2 induces c-Myc, which in turn represses
p21 and results in CDK activation and cell cycle progression. B) Antiestrogens such as
ICI repress c-Myc expression in antiestrogen-sensitive cells, thereby leading to increased
levels of p21, CDK inhibition, and cell cycle arrest. Antiestrogen resistance could arise if
c-Myc expression is no longer inhibited by antiestrogens, as this would lead to decreased
p21 levels, CDK activation, and cell cycle progression.

107

Our experiments suggest that regulation of p21 by c-Myc occurs at a
transcriptional level, since p21 protein levels, mRNA levels and promoter activity all
decrease 2-3 fold in the presence of c-Myc. They are in agreement with several reports in
other systems which show that c-Myc can repress transcription from the p21 promoter
(35-39), and offer a potential explanation for the observation that under certain conditions
only the trans-repression and not the trans-activation activity of c-Myc may be required
for cell cycle progression (57, 58). However, they differ from an earlier study in which
antisense oligonucleotides were used to decrease c-Myc expression in MCF-7 cells, and
no increases in p21 levels were observed (28). One difference between the two studies is
that the earlier report focused on early events (6—16 h) following transfection with c—myc
antisense, while we studied cells at 48 h after transfection with c-myc siRNA. We chose
this time point because our previous work and others have shown that upon ICI
treatment, p21 accumulates over 48 h leading to CDK inhibition and complete cell cycle
arrest (51, 52).

Several interesting questions are raised by our findings, including whether
additional targets of c-Myc are contributing to its ability to promote cell cycle
progression in the presence of antiestrogens. Previous studies have shown that
decreasing p21 levels using antisense oligonucleotides is sufficient to abrogate a cell
cycle arrest caused by antiestrogens (22), indicating that additional targets need not be
involved. However, another report showed that c-Myc induction in ICI-treated MCF-7
cells promoted the formation of p21-free CDK2 complexes without decreasing p21
protein levels (19), suggesting that other c-Myc targets might titrate p21 away from

CDK2 complexes. In that report, early S phase entry was observed in the c-Myc

108

inducible cells, and the authors suggested that this was due to leaky ectopic c-Myc
expression. Such leaky c-Myc expression might have led to lower p21 levels in the
absence of c-Myc induction, and subtle changes in p21 levels that were undetectable on
Western blots could have occurred upon further c-Myc induction. These changes could
be sufficient to activate CDK5 and promote cell cycle progression, since only a small
percentage of all CDK2 complexes need to be free of p21 to mediate S phase entry (16).

A second question arising from our initial results was whether deregulation of c-
Myc expression actually occurs during the acquisition of antiestrogen resistance. To
address this question, we examined the LCC9 cell line, which is an ER positive MCF-7
derivative that was selected in vivo for E2 independence and in vitro for ICI resistance
(43). We found that c-Myc expression was not decreased in ICI-treated LCC9 cells, and
its levels were comparable to E2-treated MCF-7 cells (Fig. 4). Thus, c-Myc expression is
deregulated in this in vitro model of antiestrogen resistance. Whether c-Myc
deregulation contributes to the development of antiestrogen resistant tumors or to
unresponsiveness in primary tumors remains to be determined. Though there is evidence
for aberrant c-Myc expression in breast tumors, more rigorous clinical studies designed
specifically to compare c-Myc expression in tumors before and after recurrence on
antiestrogens are required to directly correlate aberrant c-Myc expression with
antiestrogen resistance.

Additional questions concern the mechanism by which the c-myc gene is activated
in antiestrogen-resistant cells. Our results in LCC9 cells show that c-Myc deregulation
occurs at the mRNA level (Fig. 4B). Altered expression or activity of factor(s) regulating

c-myc transcription, mutations in the c-myc promoter, c-myc gene amplification,

109

 

chromosomal rearrangements or increased mRNA stability could all provide explanations
for the deregulated c-Myc expression. However, since ER binds and activates the c-myc
promoter (59, 60), deregulated c-Myc expression is likely to be the result of altered
transcriptional control. NFkB expression and activity is upregulated in LCC9 cells (61);
this could provide a mechanism for altered c-Myc expression, since NFkB is a potent
transcriptional activator of the c-myc promoter (62). Increased expression of the
nucleolar phosphoprotein nucleophosmin has also been observed in LCC9 cells (61).
Since nucleophosmin is a direct target of c-Myc (63), our results offer a potential
explanation this observation.

Previous experiments in our laboratory have established that the ability of ICI to
increase p21 protein expression is lost or attenuated in LCC9 cells (Skildum A. et al.,
unpublished observations), and the current study showed that this is a result of altered
p21 mRNA regulation (Fig. 5A). We further demonstrated that suppressing c-Myc
expression could increase p21 levels in LCC9 cells, indicating that the loss of p21
regulation is likely to be a result of constitutive c-Myc expression (Fig. 6). The fact that
p21 is highly regulated by E2/ICI in MCF-7 cells, and that disrupting this regulation by
inhibiting p21 expression with antisense oligonucleotides promotes ICI resistance, makes
it very likely that altered p21 regulation contributes to the ICI resistant phenotype of
LCC9 cells. Several other mechanisms proposed to account for antiestrogen resistance
also converge on p21. Cyclin D1 overexpression titrates p21 away from CDK2
complexes into cyclin D1/CDK4 complexes (19), and cyclin B overexpression decreases
p21 protein, but not mRNA levels (23). In addition, antisense oligonucleotides to p21 or

p27 abrogate an antiestrogen-mediated cell cycle arrest (22), and p27 deregulation

110

 

contributes to antiestrogen resistance in LY2 cells (64). Together, our results and these
reports strongly support a central role for CDK inhibitors and their direct upstream
regulators in antiestrogen resistance. Levels of these CDK inhibitors are also suppressed
in androgen-independent prostrate cancer cells (65-67), indicating that they may play a

more general role in resistance to hormonal regulation.

111

10.

REFERENCES

Systemic treatment of early breast cancer by hormonal, cytotoxic, or immune
therapy. 133 randomised trials involving 31,000 recurrences and 24,000 deaths
among 75,000 women. Early Breast Cancer Trialists' Collaborative Group.
Lancet, 339: 1-15, 1992.

Tamoxifen for early breast cancer: an overview of the randomised trials. Early
Breast Cancer Trialists' Collaborative Group. Lancet, 351: 1451-1467, 1998.

Rutqvist, L. E., Cedermark, B., Glas, U., J ohansson, H., Nordenskjold, B., Skoog,
L., Somell, A., Theve, T., Friberg, S., and Askergren, J. The Stockholm trial on
adj uvant tamoxifen in early breast cancer. Correlation between estrogen receptor
level and treatment effect. Breast Cancer Res Treat, 10: 255-266, 1987.

Wolf, D. M. and Jordan, V. C. William L. McGuire Memorial Symposium. Drug
resistance to tamoxifen during breast cancer therapy. Breast Cancer Res Treat, 27:
27-40, 1993.

Doisneau-Sixou, S. F., Sergio, C. M., Carroll, J. S., Hui, R., Musgrove, E. A., and
Sutherland, R. L. Estrogen and antiestrogen regulation of cell cycle progression in
breast cancer cells. Endocr Relat Cancer, 10: 179-186, 2003.

Leung, B. S. and Potter, A. H. Mode of estrogen action on cell proliferation in
CAMA-l cells: II. Sensitivity of G1 phase population. J Cell Biochem, 34: 213-
225, 1987.

Coezy, E., Borgna, J. L., and Rochefort, H. Tamoxifen and metabolites in MCF7
cells: correlation between binding to estrogen receptor and inhibition of cell
growth. Cancer Res, 42: 317-323, 1982.

Osborne, C. K., Boldt, D. H., Clark, G. M., and Trent, J. M. Effects of tamoxifen
on human breast cancer cell cycle kinetics: accumulation of cells in early G1
phase. Cancer Res, 43: 3583—3585, 1983.

Tsai, L. H., Lees, E., F aha, B., Harlow, E., and Riabowol, K. The cdk2 kinase is
required for the Gl-to-S transition in mammalian cells. Oncogene, 8: 1593-1602,
1993.

Kato, J ., Matsushime, H., Hiebert, S. W., Ewen, M. E., and Sherr, C. J. Direct
binding of cyclin D to the retinoblastoma gene product (pr) and pr
phosphorylation by the cyclin D-dependent kinase CDK4. Genes Dev, 7: 331-
342, 1993.

112

 

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

Sherr, C. J. G1 phase progression: cycling on cue. Cell, 79: 551-555, 1994.
Morgan, D. 0. Principles of CDK regulation. Nature, 374: 131-134, 1995.
Sherr, C. J. Mammalian G1 cyclins. Cell, 73: 1059-1065, 1993.

Sherr, C. J. and Roberts, J. M. Inhibitors of mammalian G1 cyclin-dependent
kinases. Genes Dev, 9: 1149-1163, 1995.

Altucci, L., Addeo, R., Cicatiello, L., Dauvois, S., Parker, M. G., Truss, M.,
Beato, M., Sica, V., Bresciani, F., and Weisz, A. 17beta-Estradiol induces cyclin
D1 gene transcription, p36D1-p34cdk4 complex activation and p105Rb
phosphorylation during mitogenic stimulation of G(l)-arrested human breast
cancer cells. Oncogene, 12: 2315-2324, 1996.

Prall, O. W., Sarcevic, B., Musgrove, E. A., Watts, C. K., and Sutherland, R. L.
Estrogen-induced activation of Cdk4 and Cdk2 during Gl-S phase progression is
accompanied by increased cyclin D1 expression and decreased cyclin-dependent
kinase inhibitor association with cyclin E-Cdk2. J Biol Chem, 272: 10882-10894,
1997.

Planas-Silva, M. D. and Weinberg, R. A. Estrogen-dependent cyclin E-cdk2
activation through p21 redistribution. Mol Cell Biol, 1 7: 4059-4069, 1997.

Foster, J. S., Henley, D. C., Bukovsky, A., Seth, P., and Wimalasena, J.
Multifaceted regulation of cell cycle progression by estrogen: regulation of Cdk
inhibitors and Cdc25A independent of cyclin Dl-Cdk4 function. Mol Cell Biol,
21: 794-810, 2001.

Prall, O. W., Rogan, E. M., Musgrove, E. A., Watts, C. K., and Sutherland, R. L.
c-Myc or cyclin D1 mimics estrogen effects on cyclin E-Cdk2 activation and cell
cycle reentry. Mol Cell Biol, 18: 4499-4508, 1998.

Akli, S., Zheng, P. J ., Multani, A. S., Wingate, H. F ., Pathak, S., Zhang, N.,
Tucker, S. L., Chang, S., and Keyomarsi, K. Tumor-specific low molecular
weight forms of cyclin E induce genomic instability and resistance to p21, p27,
and antiestrogens in breast cancer. Cancer Res, 64 : 3198-3208, 2004.

Bindels, E. M., Lallemand, F., Balkenende, A., Verwoerd, D., and Michalides, R.
Involvement of GI/S cyclins in estrogen-independent proliferation of estrogen
receptor-positive breast cancer cells. Oncogene, 21: 8158-8165, 2002.

Cariou, S., Donovan, J. C., Flanagan, W. M., Milic, A., Bhattacharya, N., and
Slingerland, J. M. Down-regulation ofp21WAF1/CIP1 or p27Kipl abrogates
antiestrogen-mediated cell cycle arrest in human breast cancer cells. Proc Natl
Acad Sci U S A, 97: 9042-9046, 2000.

113

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

Dhillon, N. K. and Mudryj, M. Ectopic expression of cyclin B in estrogen
responsive cells abrogates antiestrogen mediated growth arrest. Oncogene, 21:
4626-4634, 2002.

Varma, H. and Conrad, S. E. Reversal of an antiestrogen-mediated cell cycle
arrest of MCF-7 cells by viral tumor antigens requires the retinoblastoma protein-
binding domain. Oncogene, 19: 4746-4753, 2000.

Dubik, D., Dembinski, T. C., and Shiu, R. P. Stimulation of c-myc oncogene
expression associated with estrogen-induced proliferation of human breast cancer
cells. Cancer Res, 4 7: 6517-6521, 1987.

Dubik, D. and Shiu, R. P. Transcriptional regulation of c-myc oncogene
expression by estrogen in hormone-responsive human breast cancer cells. J Biol
Chem, 263: 12705-12708, 1988.

Nasi, S., Ciarapica, R., Jucker, R., Rosati, J ., and Soucek, L. Making decisions
through Myc. FEBS Lett, 490: 153-162, 2001.

Carroll, J. S., Swarbrick, A., Musgrove, E. A., and Sutherland, R. L. Mechanisms
of growth arrest by c-myc antisense oligonucleotides in MCF-7 breast cancer

cells: implications for the antiproliferative effects of antiestrogens. Cancer Res,
62: 3126-3131, 2002.

Watson, P. H., Pon, R. T., and Shiu, R. P. Inhibition of c-myc expression by
phosphorothioate antisense oligonucleotide identifies a critical role for c-myc in
the growth of human breast cancer. Cancer Res, 51 : 3996-4000, 1991.

Swarbrick, A., Akerfeldt, M. C., Lee, C. 8., Sergio, C. M., Caldon, C. E., Hunter,
L. J ., Sutherland, R. L., and Musgrove, E. A. Regulation of cyclin expression and

cell cycle progression in breast epithelial cells by the helix-loop-helix protein Idl.
Oncogene, 24: 381-389, 2005.

Wang, Y. H., Liu, S., Zhang, G., Zhou, C. Q., Zhu, H. X., Zhou, X. B., Quan, L.
P., Bai, J. F ., and Xu, N. Z. Knockdown of c-Myc expression by RNAi inhibits

MCF-7 breast tumor cells growth in vitro and in vivo. Breast Cancer Res, 7:
R220-228, 2005.

Venditti, M., Iwasiow, B., Orr, F. W., and Shiu, R. P. C-myc gene expression
alone is sufficient to confer resistance to antiestrogen in human breast cancer
cells. Int J Cancer, 99: 35-42, 2002.

Santoni-Rugiu, E., Falck, J., Mailand, N., Bartek, J ., and Lukas, J. Involvement of

Myc activity in a G(1)/S-promoting mechanism parallel to the pr/EZF pathway.
Mol Cell Biol, 20: 3497-3509, 2000.

114

 

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

Hermeking, H., Rago, C., Schuhmacher, M., Li, Q., Barrett, J. F., Obaya, A. J .,
O'Connell, B. C., Mateyak, M. K., Tam, W., Kohlhuber, F., Dang, C. V., Sedivy,
J. M., Eick, D., Vogelstein, B., and Kinzler, K. W. Identification of CDK4 as a
target of c-MYC. Proc Natl Acad Sci U S A, 97: 2229-2234, 2000.

Seoane, J ., Le, H. V., and Massague, J. Myc suppression of the p21(Cipl) Cdk
inhibitor influences the outcome of the p53 response to DNA damage. Nature,
419: 729-734, 2002.

Claassen, G. F. and Ham, S. R. A role for transcriptional repression of p21CIP1
by c-Myc in overcoming transforming growth factor beta -induced cell-cycle
arrest. Proc Natl Acad Sci U S A, 97: 9498-9503, 2000.

Gartel, A. L., Ye, X., Goufrnan, E., Shianov, P., Hay, N., Najmabadi, F., and
Tyner, A. L. Myc represses the p21(WAF1/CIPl) promoter and interacts with
Spl/Sp3. Proc Natl Acad Sci U S A, 98: 4510-4515, 2001.

Mitchell, K. O. and El-Deiry, W. S. Overexpression of c-Myc inhibits
p21WAF1/CIP1 expression and induces S-phase entry in 12-0-
tetradecanoylphorbol-l3-acetate (TPA)-sensitive human cancer cells. Cell Growth
Differ, 10: 223-230, 1999.

Wu, 8., Cetinkaya, C., Munoz-Alonso, M. J ., von der Lehr, N., Bahrarn, F.,
Beuger, V., Eilers, M., Leon, J ., and Larsson, L. G. Myc represses differentiation-
induced p21CIP1 expression via Miz-l-dependent interaction with the p21 core
promoter. Oncogene, 22: 351-360, 2003.

Yang, W., Sherr, J., Wu, M., Arsura, M., FitzGerald, M., Suldan, 2., Kim, D. W.,
Hofrnann, C. S., Pianetti, S., Romieu-Mourez, R., Freedman, L. P., and
Sonenshein, G. E. Repression of transcription of the p27(Kip1) cyclin-dependent
kinase inhibitor gene by c-Myc. Oncogene, 20: 1688-1702, 2001.

Shafie, S. M. and Grantham, F. H. Role of hormones in the growth and regression
of human breast cancer cells (MCF-7) transplanted into athymic nude mice. J Natl
Cancer Inst, 67: 51-56, 1981.

Soule, H. D., Vazguez, J ., Long, A., Albert, S., and Brennan, M. A human cell
line from a pleural effusion derived fiom a breast carcinoma. J Natl Cancer Inst,
51: 1409-1416, 1973.

Brunner, N., Boysen, B., Jirus, S., Skaar, T. C., Holst-Hansen, C., Lippman, J .,
Frandsen, T., Spang-Thomsen, M., Fuqua, S. A., and Clarke, R. MCF7/LCC9: an
antiestrogen-resistant MCF-7 variant in which acquired resistance to the steroidal
antiestrogen ICI 182,780 confers an early cross-resistance to the nonsteroidal
antiestrogen tamoxifen. Cancer Res, 57: 3486-3493, 1997.

115

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

Mai, S. Overexpression of c-myc precedes amplification of the gene encoding
dihydrofolate reductase. Gene, 148: 253-260, 1994.

Olson, L. K., Qian, J ., and Poitout, V. Glucose rapidly and reversibly decreases
IN S-l cell insulin gene transcription via decrements in STF-l and C1 activator
transcription factor activity. Mol Endocrinol, 12: 207 -2 1 9, 1998.

Blackwood, E. M., Lugo, T. G., Kretzner, L., King, M. W., Street, A. J ., Witte, O.
N., and Eisenman, R. N. Functional analysis of the AUG- and CUG-initiated
forms of the c-Myc protein. Mol Biol Cell, 5: 597-609, 1994.

Datto, M. B., Yu, Y., and Wang, X. F. Functional analysis of the transforming
grth factor beta responsive elements in the WAF 1/Cip1/p21 promoter. J Biol
Chem, 270: 28623-28628, 1995.

Matsushime, H., Quelle, D. E., Shurtleff, S. A., Shibuya, M., Sherr, C. J ., and
Kato, J. Y. D-type cyclin-dependent kinase activity in mammalian cells. Mol Cell
Biol, 14: 2066-2076, 1994.

Evan, G. 1., Lewis, G. K., Ramsay, G., and Bishop, J. M. Isolation of monoclonal

antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol, 5:
3610-3616, 1985.

Zindy, F., Lamas, E., Chenivesse, X., Sobczak, J ., Wang, J ., F esquet, D.,
Henglein, B., and Brechot, C. Cyclin A is required in S phase in normal epithelial
cells. Biochem Biophys Res Commun, 182: 1144-1154, 1992.

Skildum, A. J ., Mukherjee, S., and Conrad, S. E. The cyclin-dependent kinase
inhibitor p21WAF1/Cip1 is an antiestrogen-regulated inhibitor of Cdk4 in human
breast cancer cells. J Biol Chem, 277: 5145-5152, 2002.

Carroll, J. S., Prall, O. W., Musgrove, E. A., and Sutherland, R. L. A pure
estrogen antagonist inhibits cyclin E-Cdk2 activity in MCF-7 breast cancer cells

and induces accumulation of p130-E2F4 complexes characteristic of quiescence. J
Biol Chem, 275: 38221-38229, 2000.

Prall, O. W., Carroll, J. S., and Sutherland, R. L. A low abundance pool of nascent
p21WAF1/Cip1 is targeted by estrogen to activate cyclin E*Cdk2. J Biol Chem,
276: 45433-45442, 2001.

Mittnacht, S. and Weinberg, R. A. Gl/S phosphorylation of the retinoblastoma
protein is associated with an altered affinity for the nuclear compartment. Cell,
65: 381-393, 1991.

116

 

 

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

Lippman, M., Bolan, G., and Huff, K. The effects of estrogens and antiestrogens
on hormone-responsive human breast cancer in long-term tissue culture. Cancer
Res, 36: 4595-4601, 1976.

Osborne, C. K., Boldt, D. H., and Estrada, P. Human breast cancer cell cycle

synchronization by estrogens and antiestrogens in culture. Cancer Res, 44: 1433-
1439, 1984.

Spotts, G. D., Patel, S. V., Xiao, Q., and Hann, S. R. Identification of
downstream-initiated c-Myc proteins which are dominant-negative inhibitors of
transactivation by full-length c-Myc proteins. Mol Cell Biol, 1 7: 1459-1468,
1997.

Xiao, Q., Claassen, G., Shi, J ., Adachi, S., Sedivy, J ., and Hann, S. R.
Transactivation-defective c-MycS retains the ability to regulate proliferation and
apoptosis. Genes Dev, 12: 3803-3808, 1998.

Dubik, D. and Shiu, R. P. Mechanism of estrogen activation of c-myc oncogene
expression. Oncogene, 7: 1587-1594, 1992.

Shang, Y. and Brown, M. Molecular determinants for the tissue specificity of
SERMs. Science, 295: 2465-2468, 2002.

Gu, Z., Lee, R. Y., Skaar, T. C., Bouker, K. B., Welch, J. N., Lu, J ., Liu, A., Zhu,
Y., Davis, N., Leonessa, F., Brunner, N., Wang, Y., and Clarke, R. Association of
interferon regulatory factor-l , nucleophosmin, nuclear factor-kappaB, and cyclic
AMP response element binding with acquired resistance to F aslodex (ICI
182,780). Cancer Res, 62: 3428-3437, 2002.

La Rosa, F. A., Pierce, J. W., and Sonenshein, G. E. Differential regulation of the

c-myc oncogene promoter by the NF-kappa B rel family of transcription factors.
Mol Cell Biol, 14: 1039-1044, 1994.

Zeller, K. I., Haggerty, T. J ., Barrett, J. F., Guo, Q., Wonsey, D. R., and Dang, C.
V. Characterization of nucleophosmin (B23) as a Myc target by scanning
chromatin immunoprecipitation. J Biol Chem, 276: 48285-48291, 2001.

Donovan, J. C., Milic, A., and Slingerland, J. M. Constitutive MEK/MAPK
activation leads to p27(Kip1) deregulation and antiestrogen resistance in human
breast cancer cells. J Biol Chem, 276: 40888-40895, 2001.

Knight-Krajewski, S., Welsh, C. F ., Liu, Y., Lyons, L. S., Faysal, J. M., Yang, E.
S., and Bumstein, K. L. Deregulation of the Rho GTPase, Racl, suppresses
cyclin-dependent kinase inhibitor p21(CIP1) levels in androgen-independent
human prostate cancer cells. Oncogene, 23: 5513-5522, 2004.

117

66.

67.

Zhuang, S. H. and Bumstein, K. L. Antiproliferative effect of 1alpha,25-
dihydroxyvitamin D3 in human prostate cancer cell line LNCaP involves

reduction of cyclin-dependent kinase 2 activity and persistent G1 accumulation.
Endocrinology, 139: 1197-1207, 1998.

Graff, J. R., Konicek, B. W., McNulty, A. M., Wang, Z., Houck, K., Allen, S.,
Paul, J. D., Hbaiu, A., Goode, R. G., Sandusky, G. E., Vessella, R. L., and
Neubauer, B. L. Increased AKT activity contributes to prostate cancer progression
by dramatically accelerating prostate tumor growth and diminishing p27Kip1
expression. J Biol Chem, 275 : 24500-24505, 2000.

118

   

lllllllllllulllillilllllll