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THE ROLE OF RETINOBLASTOMA PROTEIN AND
INSULIN LIKE GROWTH FACTOR SIGNALING IN

ANTI ESTROGEN RES I STANCE

presented by

Hemant Varma

has been accepted towards fulfillment
of the requirements for

Ph . D. degree inBiochemistry
and Molecular Biology

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THE ROLE OF RETINOBLASTOMA PROTEIN AND INSULIN LIKE GROWTH
FACTOR SIGNALING IN ANT IESTROGEN RESISTANCE

By

Hemant Varma

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Biochemistry and Molecular Biology

2001

ABSTRACT

THE ROLE OF RETINOBLASTOMA PROTEIN AND INSULIN LH(E GROWTH
FACTOR SIGNALING IN ANTIESTROGEN RESISTANCE

By

Hemant Varma

Approximately 40% of all human breast cancers are estrogen dependent for
proliferation, making them responsive to antiestrogen therapy. However during the
disease course, these tumors often progress to an antiestrogen resistant phenotype,
resulting in high mortality and underscoring an urgent need to understand the basis of
antiestrogen resistance. Members of the retinoblastoma family proteins are key negative
regulators of cell cycle progression and are frequently mutated in a variety of tumors
including breast tumors. MCF-7 is a human breast cancer cell line, that proliferates in
response to estrogen treatment and is growth inhibited upon treatment with antiestrogens.
The results presented in this thesis demonstrate that inactivation of the tumor suppressors
of the retinoblastoma family using DNA tumor virus large T antigens enables MCF-7
cells to proliferate in the presence of antiestrogens. Using a number of SV4O large T
mutants, we further demonstrate that the inactivation of pr family members by large T
is absolutely required for and may be sufficient for MCF—7 cells to acquire antiestrogen
resistance. In addition, we have established MCF—7 derivatives that inducibly express

Polyoma large T and find that expression of large T in antiestrogen treated MCF-7 cells

causes an induction of cyclin A, cdk2 and cdk4 activities, key regulators of cell cycle
progression that are inhibited by antiestrogens.

In a related study, we tested whether the upregulation of Insulin Like Growth
Factor-1 (IGF-l) signaling could cause antiestrogen resistance. The impetus for this study
was the observation that IGF-l is a potent mitogen of breast cancer cells and
antiestrogens have been reported to inhibit IGF-l stimulated proliferation. Numerous
studies support the view that antiestrogens regulate breast cancer proliferation indirectly
by downregulating the IGF-l signaling pathway. Our results point to the contrary, since
antiestrogen treatment did not prevent or decrease the signaling events induced by IGF-l
including the activation of MAPK and P13K, key downstream effectors of IGF-I, as well
as some of the cell cycle changes induced by IGF-l. Further, activation of intermediates
in IGF—l signaling pathway did not confer antiestrogen resistance to MCF-7 cells. These
results suggest that activation of the IGF-l signaling cascade alone is not sufficient to
cause antiestrogen resistance.

In summary, the results of this study demonstrate that estrogen mediates its
proliferative effects in MCF-7 cells by inactivating the pr family members and suggest
that loss of function or deregulation of the pr pathway can potentially lead to
antiestrogen resistance in breast cancer patients. Additionally, estrogen affects breast
cancer proliferation without regulating growth factor (IGF—l) signaling but by impinging
on the cell cycle components. These results suggest that deregulation of IGF-I signaling

alone does not confer antiestrogen resistance in breast cancer patients.

ACKNOWLEDGEMENTS

I would like to thank my mentor Susan Conrad for her advice, support and
encouragement throughout these years. I would also like to thank my guidance committee
members, Zachary Burton, Donald Jump, Richard Miksicek and Steve Triezenberg for
their helpful advice and discussions. I would like to thank Michelle Fluck for her
encouragement through these years and to Ron Patterson for teaching me how to play the
“game”. A big thanks to the current members of Conrad lab, Shibani Mukherjee, Andy
Skildum and Yu Ping Tang for their help and their patience with high entropy.

Also thanks to all the past members of our lab and the Fluck lab, Feng Han,
Heather Hirsch, Christopher Ontiveros, Maria Pino and Cunjie Zhang for their help and
friendship over the years. I would also like to thank Sridhar Venkataraman and Dipnath
Baidyaroy for their friendship, encouragement and for sharing their enthusiasm for

science.

iv

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

CHAPTER 1

CHAPTER 2

Introduction

Mammary Gland Development

Breast Cancer Genetics

Estrogen, Antiestrogens and Breast Cancer
Estrogen Receptor: Mechanism of action of estrogen
Eukaryotic Cell Cycle

Tumor Suppressors of the pr family

Kinase Inhibitory Proteins: KIP and INK4 Families
Cdks, Cyclins, and Cdc25 phosphatases

Estrogen and cell cycle regulation

Growth Factors and Breast Cancer

Antiestrogen Therapy and Antiestrogen Resistance
References

vii

viii

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16
19
23
28

Reversal of an Antiestrogen-Mediated Cell Cycle Arrest of MCF-7 Cells by Viral Tumor
Antigens Requires the Retinoblastoma Protein-Binding Domain

CHAPTER 3

Abstract

Introduction

Materials and Methods
Results

Discussion

References

51
52
55
58
69
74

Abrogation of Polyoma LT Induced Proliferation of Antiestrogen Treated MCF-7 Cells
by p21wan but not by pl6INK4A

Abstract

Introduction

Materials and Methods
Results

Discussion

References

81
82
85
90
103
109

CHAPTER 4

Antiestrogen ICI 182,780 Decreases Proliferation of IGF-I Treated MCF-7 Cells Without
Inhibiting the PI3K/AKT Pathway.

Abstract 1 13
Introduction 1 14
Materials and Methods 116
Results 121
Discussion 134
References 1 39

CHAPTER 5

Summary and Future Experiments 148

vi

LIST OF TABLES

Table 2.1. Characteristics of wild type and mutant viral T-antigens used in study. 59

vii

11;

LIST OF FIGURES

Figure 1.1. Simplified model of the eukaryotic cell cycle. 7

Figure 1.2 Model of cyclin/Cdk pathway. 10

Figure 1.3. Interaction of IGF-I and estrogen signaling. 22

Figure 2.1. Model for pr inactivation by estrogen treatment and LT expression in 54
MCF-7 cells.

Figure 2.2. Ability of wild type and mutant LT proteins to promote antiestrogen 62
reswtance.

Figure 2.3 Double indirect immunofluorescence of transfected cells. 63

Figure 2.4 Effects of PyLT and dominant negative p53 expression on ICI 66

sensitivity in MCF-7 cells.

Figure 2.5 Effects of PyLT expression on ICI sensitivity in stably transfected cells. 67
Figure 3.1 Models for the regulation of Cdk4 and Cdk2 activities by antiestrogens. 84

Figure 3.2 MCF—7 cells expressing PyLT are sensitive to p21wan but refractory to 92
p161NK4A arrest.

Figure 3.3 Selection of stable cell lines that express PyLT in an inducible manner. 94

Figure 3.4 PyLT induces S phase entry in ICI treated MCF-7 cells in short term 96

assays.
Figure 3.5 PyLT induces proliferation in ICI treated cells over 2 cell cycles. 99
Figure 3.6 PyLT induces Cdk2 and Cdk4 activities in ICI treated MCF—7 cells. 100
Figure 3.7 PyLT confers partial antiestrogen resistance in long term assays. 102

Figure 3.8 Model for the regulation of Cdk4 and Cdk2 activities by antiestrogen. 108
Figure 4.1 ICI decreases proliferation in the presence of IGF-I. 122

Figure 4.2 ICI pre-treatment does not abrogate IGF-l induced expression 125
of cyclin D1 or proliferation.

Figure 4.3 ICI pre-treatment does not prevent induction of cyclin D1 mRNA 127
by IGF-l.

viii

Figure 4.4 IGF-l induced PI3K activation is required for cyclin D1 expression, 129
and is inhibited by LY294002 but not by ICI.

Figure 4.5 Constitutively active P13K or AKT are insufficient to overcome an ICI 133
mediated cell cycle arrest.

Figure 4.6 Model of IGF-1 and ER signaling interaction. 138

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CHAPTER 1

INTRODUCTION

Mammary Gland Development

Mammalian newborns are dependent on maternal nutrition in the form of milk for
survival during early infancy. To nurse their young ones mammals have evolved
specialized organs called mammary glands that produce and secrete milk. The mammary
glands remain rudimentary until puberty; they then develop in preparation for
reproduction and eventual nursing of the newborn. At puberty, breast ducts grow and
show increased branching, in part due to epithelial cell proliferation in response to
complex hormonal and growth factor stimulation (1). This sets the stage for later
development of lobuloalveoli, the milk producing tissue at the ends of ducts, which
occurs during pregnancy.

During the menstrual cycle, cyclic changes in the levels of hormones cause
alterations in breast epithelial cell proliferation (2). These regular alterations in the
proliferative status of breast epithelial tissue during the long reproductive span (30—40
years) of women, makes breast epithelial tissue highly susceptible to tumor formation. At
least one in eight women in the US. will develop breast cancer during her lifetime
making it the most common cancer among women with 90 percent of these tumors being
epithelial in origin. Despite current therapies, 50 percent of women diagnosed with breast

cancer eventually die of the disease, making it the second leading cause of cancer death

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among women in the USA. The high incidence and mortality associated with breast

cancer underscores the need for improved therapy for this disease.

Breast Cancer Genetics

Aberrant gene expression has been implicated in the development of breast cancer
with the identification of genes whose deregulation can cause increased susceptibility to
the genesis of breast cancer. Many insights have arisen from transgenic mouse models,
where specific gene products are overexpressed in the mammary gland using mammary
specific promoters. This has led to the identification of a number of genes including
erbB2/Neu, c-myc, Wnt-l and cyclin D1 whose overexpression can potentially lead to the
development of breast cancers (3-7). On the other hand, familial cases of breast cancer
have led to the identification of two genes, BRCA1 and BRCA2, that act as tumor
suppressors and whose loss leads to the development of breast cancer at a very high
frequency (8). Mutations in BRCA1 and BRCA2 account for only 5-10 percent of breast

tumors (9).

Estrogen /Antiestrogens and Breast Cancer

Despite the identification of genes responsible for breast cancer in a small subset
of patients, current knowledge regarding the genetic alterations in breast cancer is
insufficient to target specific therapy for a large majority of patients. Furthermore, the
underlying genetic changes leading to breast cancer are likely to vary widely between
patients, preventing any generalized therapy. However, the proliferation of a large

percentage of breast tumor cells remains responsive to estrogens, and the role of estrogen

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in breast cancer growth is highlighted by the fact that 40% of breast cancers require
estrogen for proliferation (10). The first insight into the regulation of breast cancer
proliferation by estrogen was provided by Beatson’s observation in the 1890’s that
surgical removal of the ovaries of breast cancer patients caused tumor regression (11). It
was later discovered that estrogens were key ovarian hormones involved in both normal
and breast tumor proliferation. Estrogens are steroidal compounds that play a role in
regulating the development, differentiation and proliferation of reproductive tissues. 17(3-
estradiol, esterone and estriol are the major estrogenic compounds secreted by ovaries,
and of these 17B-estradiol is the most potent estrogen. The circulating levels of these
hormones increase dramatically at puberty, oscillate at these elevated levels during the
menstrual cycle throughout the reproductive span of females, and finally decrease at
menopause to prepubertal levels. These changes in the levels of estrogens correlate well
with breast epithelial proliferation, as demonstrated by studies that ovariectomy in the
prepubertal stage in mice prevents breast development and this can be reversed by
estrogen supplementation (12). Furthermore, postmenopausal women show a decrease in
ductal branching and atrophy of ducts due to decreased ovarian function.

The recognition of a key role for estrogens in breast cancer proliferation provided
the rationale for the development of antiestrogenic compounds for the treatment of breast
cancer patients. Thus, one of the major therapies for breast cancer consists of estrogen
antagonists such as Tamoxifen and ICI 182,780. Tamoxifen, a non-steroidal analogue of
estradiol is the most widely used antiestrogen therapy and has decreased breast cancer
morbidity greatly, while having minimal side effects. Despite tumor regression and

increased disease free survival, tumors initially sensitive to Tamoxifen eventually

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develop antiestrogen resistance, and continue to grow upon Tamoxifen treatment (13). In
order to develop improved therapeutics, it is essential to understand the mechanism(s) by
which estrogens regulate the proliferation of breast tumors, and how tumors evolve to an

antiestrogen resistant phenotype.

Estrogen Receptor: Mechanism of action of estrogen

Estrogens mediate their cellular effects by binding to specific receptor proteins,
estrogen receptors (ERs) that are members of a family of ligand activated nuclear
transcription factors. These are evolutionarily conserved proteins that bear homology
across vertebrates -from fish to mammals (14). Presently, two genes for ER have been
identified, Ech and ERB. Both genes encode proteins with a high degree of homology,
but with differences in tissue expression and function (15). ERor is expressed in a broad
range of tissues, whereas ERB is expressed largely in ovaries, prostate, epididymis, lung,
and hypothalamus (16). The phenotypes of ER knockout mice suggest that ER01 is
involved in breast development and proliferation, since ERor knockout mice fail to
develop breast tissue at puberty and breast tissue remains immature (17). In contrast,
ERB mice are fertile and their breast development is not impaired, suggesting that this
receptor may not play a major role in breast proliferation (18).

ERor is a multidomain nuclear phosphoprotein that regulates transcription of
cellular genes by binding to specific DNA sequences called enhancer elements present in
target genes. It is composed of a DNA binding domain, a ligand binding domain, two
transcription activation domains, one at the N-terminus (AF-1) and the other at the C-

terrninus (AF-2) and a receptor dimerization domain. At least two different modes of ER

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activation are known, one involving ligand binding and the other being ligand
independent. Ligand binding induces ER dimerization and binding to enhancer elements
called estrogen response elements (EREs) in the target genes, and induces transcription
from target genes. The C-terminal AF-2 is largely involved in this ligand induced ER
activation (19). Alternatively, the ligand bound ER may activate certain genes by protein-
protein interactions with members of the activator protein-1 (AP-l) family, and thereby
regulate the transcription of genes containing the AP—l DNA binding site (20). In
addition, ER has been reported to enhance transcription of target genes via a ligand
independent mechanism. This involves phosphorylation of certain serine residues
(ser118) in the AF-l region by growth factor signaling cascades, which enhance ER’s
transcriptional activity on ERE containing genes (21).

Ultimately, the regulation of gene transcription by ER involves recruitment of
coactivators that activate the transcriptional machinery on the promoters of target genes.
A number of coactivators have been discovered, and their roles in ER mediated
transcription have been somewhat defined. Upon ligand binding to ER, a conformational
change is induced in the AF-2 domain (22). This results in the recruitment of the p160
family of coactivators, that include SRC-l, TIP-2 or Gripl and AIB (23-25) and
associated factors involved in chromatin remodeling to the promoters. These changes
lead to the activation of RNA polymerase mediated transcription from the target genes
(26). The relevance of coactivators to ER function, as well as the overlap in coactivator
function, is demonstrated by the phenotype of SRC-l knockout mice, which are viable
and fertile, but partially resistant to the effects of estrogen and show decreased mammary

gland proliferation (27). Since coactivators are involved in ER’s actions at the

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transcriptional level, alterations of these cofactors could potentially be involved in the
deregulation of ER mediated gene expression and proliferation. Indeed, the SRC family
member AIB-1/SRC-3 is overexpressed in 60% of primary breast cancers, and is

amplified in 10% of breast tumors (24, 28).

Eukaryotic Cell Cycle

In order to increase the proliferation of cells, mitogens including estrogen have to
impinge on the machinery controlling cell proliferation. Over the past two decades, a
better understanding of the players involved in cell cycle progression and their roles in
proliferation has been gained. Cell proliferation involves the doubling of the DNA
content of a cell by the process of DNA replication, followed by equal distribution of the
DNA to daughter cells during cell division. Most somatic cells proliferate via a highly
ordered sequence of events that can be divided into four different phases. Initially cells
with a 2N content of DNA go through a growth phase called gapl or G]. During this
phase the cell integrates various internal and environmental signals before committing to
cell division. This phase is mitogen responsive, and at a point late in G1 called the
restriction point, a cell commits to cell division. Once the decision to divide is made, the
cells are mitogen insensitive and progress into S phase where DNA replication occurs.
This is followed by a gap2 or G2 phase where cells prepare for mitosis, and finally cell

division takes place by mitosis followed by cytokinesis in the M phase (See Figure 1.1).

 

 

 

 

 

 

 

 

 

 

 

 

   
 

 

 

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Figure 1.1. Simplified model of the eukaryotic cell cycle. Cells progress through a
highly ordered sequence of events called the cell cycle they replicate their DNA and
undergo cell division. Cells in G1 are stimulated by mitogens such as estrogen (E), which
enhance progression through this phase of the cell cycle. Mitogens enhance cell cycle
progression through the G1 phase by activating serine threonine kinases called Cdks of
which there are two types, Cdk4 and Cdk2. Cdk4 is activated upon binding to cyclin D in
early G1 while Cdk2 is activated as complexes with both cyclin B or cyclin A in late G1.
These kinases phosphorlylate and inactivate the pr family of tumor suppressors during
mid to late G1 that results in the release of transcription factors that regulate genes
required for DNA replication. Thereafter the cells progress into S phase where they
duplicate their DNA (from 2n to 4n). This is followed by a gap phase G2 that precedes
cell division in M Phase.

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Tumor Suppressors of the pr family

The decision to divide is based on a balance between negative and positive
regulators of cell division. At the center of the negative regulation of the cell cycle is a
family of tumor suppressors called the retinoblastoma family of proteins, which consists
of pr and the related farmly members p107 and p130. The role of pr as a tumor
suppressor was established by the fact that individuals lacking one copy of the pr gene
developed a rare form of retinal tumor called retinoblastoma (29). Further studies
demonstrated that pr serves a general role as a regulator of cell cycle progression (See
Figure 1.2) (30) and have better defined the role of pr in cell cycle progression. pr is
a 928 amino acid nuclear phosphoprotein that has at least 16 potential serine/threonine
phosphorylation sites (31, 32). In its under/hypophosphorylated state, pr binds to and
inhibits the E2F family of transcription factors (33) that regulate transcription of genes
that are important for DNA synthesis (34). Upon phosphorylation, pr is inactivated and
is no longer able to bind to the E2F family proteins, making them free to activate
transcription (35, 36). Since pr phosphorylation occurs during mid to late G1, it has
been suggested that it may represent the “restriction point” that commits cells to progress
to S phase, and that once pr is inactivated cells may be mitogen independent. The
importance of pr inactivation in cell cycle progression is highlighted by the fact that
mutations in pr are reported in a variety of tumors, including breast tumors (30).
Additionally, various DNA tumor viruses that usurp the cellular DNA replication
machinery to replicate their DNA, encode viral proteins that specifically target and
inactivate the pr family of proteins, thus inducing resting cells to initiate DNA

replication (37). Large Tumor (LT) antigens encoded by the SV40 and polyoma viruses,

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adenoviral ElA protein and E7 protein of human papillomaviruses, all bind to and
inactivate hypophosphorylated pr family members (38, 39). The role of the other pr
family members in cell cycle progression is less well defined. Although overexpression
of p107 or p130 causes growth arrest in cells, they are only rarely mutated in tumors.
Both p107 and p130 are phosphorylated in a manner similar to pr and bind to members
of the E2F family of transcription factors. The phenotype of knockout mice for the
various family members demonstrate that pr is essential for murine development, as Rb
null mice die in utero due to defects in hematogenesis and neurogenesis (40—42). In
contrast single p107 and p130 knockout mice are viable, and do not show any apparent
phenotype (43, 44) whereas p107/p130 double knockout die shortly after birth (44).
These studies highlight the essential but distinct functions of pr and p107/p130
proteins. Further, these results suggest that p107/p130 proteins have overlapping
function(s) that are distinct from pr (43, 45). Studies using mouse embryo fibroblasts
derived from Rb knockout mice show a distinct decrease in the duration of G1, and
upregulation of E2F target genes, though cells are not transformed (46). Recently, triple
knockout mice have been generated in which all three pr members are deleted, and
fibroblasts derived from these embryos show a faster population doubling time compared
to single or double knockout mice with any combination of pr family members
inactivated. Further, these cells are not contact inhibited, are immortalized in culture, and
show features of transformed cells. This provides strong evidence that the loss of all three
members contributes to transformation, and suggests some degree of functional overlap

between the pr family members (47).

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1674 Fig.2). In this model cyclin D1/Cdk4 complexes phosphorylate the pr family
thereby releasing the E2F family of transcription factors. E2F regulated genes such as
cyclin B and cyclin A get induced and activate Cdk2 to promote S phase entry. A positive
feedback loop exists wherein the activated Cdk2 complexes can also phosphorylate pr.
Cyclin-Cdk activity is also controlled b interaction with two families of Cdk inhibitors:
the INK4 family which includes p16IN 4“, and the p21W'/p27""’ family. An additional
level of regulation is provided by Cdc25A phoshatase, which removes inhibitory
phosphorylation on Cdk’s, and by the Cdk activating kinases (CAKs), which carry out
phosphorylation/dephosphorylation of specific conserved residues on the Cdks. Some of
the known targets of estrogen are indicated.

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Kinase Inhibitory Proteins: KIP and INK4 Families

A second class of tumor suppressors consists of two families of cyclin dependent
kinase inhibitors, the INK4 and the KIP families, which negatively regulate progression
through the cell cycle. The INK4 family of kinase inhibitors consists of four proteins
named for their apparent molecular weight of p15, p16, p18 and p19 (48). These proteins,
of which p16INK4A was initially discovered, bind to and inactivate a subset of kinases,
Cdk4/6, that phosphorylate pr (49). The members of the INK4 family prevent cell cycle
progression by inhibiting the activity of kinases involved in pr phosphorylation, as
demonstrated by their ability to inhibit proliferation in the presence but not in the absence

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the observation that p1 is frequently mutated in tumors and the fact that p1

knockout mice though viable, have an increased frequency of tumors (51). The role of the
other family members in vivo appears to be similar to that of p16INK4A.

The second family consists of three kinase inhibitors, p21wa“, p27Kip' and p57Kip2.
These kinase inhibitors bind to and inhibit Cdk2 activity (52). Their role as inhibitors of
Cdk4/6 is more controversial, since some reports suggest that they are inhibitors of
Cdk4/6 (52) while others have reported that these are necessary for Cdk4/6 complex
formation (53). The levels of these kinase inhibitors are regulated by mitogens and other
growth stimuli, with mitogens downregulating and anti-mitogens increasing their levels.
This is demonstrated by contact and TGFB mediated arrest of mink lung fibroblasts,

which is caused by increases in p27Kipl protein levels and resulting decreases in Cdk2

activity (54). Consistent with its role as a tumor suppressor, p21wan knockout mice have

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defective G1 checkpoint controls (55). A similar phenotype was noted for p27mpl
knockout mice, with increased body weight, tumor formation and increased Cdk2 activity
(56, 57). The p57Kipz knockout mice have severe developmental abnormalities (58),
suggesting functional differences between the various Kip family members. Interestingly,
double knockout mice generated by knocking out individual members from the two
different families, Kip and INK4, show enhanced tumor formation compared to either
alone, suggesting functional collaboration between the different families of cyclin-

dependent kinase inhibitors (59).

Cdks, Cyclins , and Cdc25 phosphatases

The positive regulators of the cell cycle are a group of serine/threonine kinases“
that in the active state exist as binary complexes of a catalytic and a regulatory subunit
(60). The catalytic unit is a cyclin dependent kinase (Cdk) of which there are two major
types Cdk4/6 and Cdk2 in higher eukaryotes. The levels of expression of the Cdks are
constitutive, but their activity is regulated by multiple mechanisms including by binding
to specific cyclin partners, interaction with two families of Cdk inhibitors,
phosphorylation on certain conserved residues by Cdk activating enzymes and
dephosphorylation by phosphatases of the Cdc25A family. The levels of cyclins are
tightly regulated by mitogenic stimuli and the cell cycle phase. By regulating the levels of
specific cyclins, mitogens and antimitogens regulate the activities of specific Cdks during
the different phases of the cell cycle.

Cyclin D is a major group of G1 cyclins, consisting of three different proteins D1,

D2 and D3 with overlapping expression in proliferating cells. These proteins are

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expressed in response to mitogen stimulation, and activate the Cdk4/6 group of cyclin
dependent kinases (61). The only known targets for cyclin D-Cdk4/6 complexes are the
members of the pr protein family (62). Cyclin D1 is better studied compared to the
other members of the cyclin D family and appears to play an important role in the
proliferation of both normal and tumor breast tissue. The role of cyclin D1 protein in
mediating mitogenic responses is highlighted by the fact that overexpression of cyclin D1
reduces serum dependence for proliferation and accelerates G1 progression (63-65).
Further, overexpression of cyclin D1 is found in a large percentage of tumors including
breast cancers (30, 66, 67). Transgenic mice overexpressing cyclin D1 under a mammary
specific promoter develop mammary carcinomas (7). Conversely, cyclin D1 knockout
mice are smaller in size than wild type mice, show defects in mammary gland
development, and are resistant to tumor formation in response to overexpression of
certain oncogenes showing an important and somewhat specific role for cyclin D1 in
mammary tissue proliferation (68). Recently, a role for cyclin D1-Cdk4 complexes in
sequestering p21wm and p27Kipl from the Cdk2 complexes has been proposed (69). This
model is supported by in vivo genetic evidence, demonstrating that the phenotype of
cyclin D1 knockout mice is restored in p27mpllcyclin D1 double knockout mice (70).
Cyclin E, the other major G1 cyclin, forms complexes with and activates Cdk2
and plays an essential role in G1 and S phase progression (71-73). Cyclin E is
upregulated in response to mitogenic stimuli and has E2F responsive elements in its
promoter, making it a downstream target of pr (74). One of the targets of cyclin E/Cdk2
complexes are the pr family members, suggesting the presence of a feedback loop that

enhances the inactivation of pr and accumulation of active cyclin E-Cdk2 kinase

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complexes (Figure 1.2) (74). Evidence in the past few years has suggested that cyclin B-
Cdk2 may be the ultimate activity required for GI progression and entry into S phase.
Cyclin D1 knockout mice recover their phenotype upon expression of cyclin E in target
tissues (75). Further, growth arrest mediated via activation of pr using a constitutively
active pr that is non-phosphorylatable (76) or by inhibition of Cdk4/6 activities by
p16INK4A can be overcome by cyclin E overexpression, placing cyclin E-Cdk2 kinase
activity downstream of cyclin D1-Cdk4 and pr (77). The cyclin E-Cdk2 has targets
other than pr family members, as inferred by its requirement for proliferation in pr
negative cells, unlike cyclin D1 which is dispensable in such cells (73). Recently, several
novel substrates of cyclin E-Cdk2 have been identified, including NPAT, a
transcriptional activator of histone genes. Histones are essential for the packaging of
DNA into nucleosomes, partly explaining the importance of cyclin E-Cdk2 for
proliferation (78). Although all the targets of cyclin E-Cdk2 are yet to be identified, it is
clear that its kinase activity is essential for G1 progression and S phase entry, probably by
a pathway parallel to or downstream of pr.

An additional layer of regulation of Cdks occurs at the post-translational level by
phosphorylation and dephosphorylation events. Cdks are phosphorylated at a conserved
threonine residue by Cdk activating kinase (CAK), with phosphorylation at this site being
required for Cdk activation (60). Cdk2 activation also involves the removal of a
deactivating phosphorylation that is accomplished by a phosphatase called Cdc25A.
Cdc25A is a member of Cdc25 family of dual-specificity phosphatases that

dephosphorylates the negative regulatory phosphates at threonine-14 and tyrosine-15 in

Cdks, leading to their activation (79). The Cdc25 family of phosphatases consists of three

14

 

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different enzymes Cdc25A, B and C, encoded by 3 different genes. Of these, Cdc25A
phosphatase is involved in the Gl-S transition, since antisense to Cdc25A arrests cells in
the G] phase of the cell cycle (80) and overexpression leads to premature activation of
Cdk2 activity, and decreased duration of G1 (81). Moreover, overexpression of Cdc25A
has been found in variety of tumors implicating it as an oncogene (82). Interestingly,
Cdc25A activity is regulated by phosphorylation that is accomplished by cyclin E-Cdk2
kinase (83) suggesting a positive feedback loop whereby the cyclin E-Cdk2 activates
Cdc25A that in turn activates cyclin E-Cdk2 by dephosphorylation of Cdk2. In addition,
reports suggest that Cdk4 activity may be activated by Cdc25A mediated de-
phosphorylation under certain circumstances (84).

Once cells have committed to DNA replication in late G1 phase, they enter a
phase of duplication of the DNA content called S phase. Entry into and progression
through this phase requires cyclin A-Cdk2 activity that is induced at the Gl-S phase
boundary and is highly expressed throughout S phase. Inhibition of cyclin A by
microinjecting antibodies to cyclin A results in an arrest at the Gl-S boundary (85). The
cyclin A gene is positively regulated by E2F transcription factors, and is induced as a
consequence of pr inactivation.

After DNA replication in S phase, cells pass through a G2 or growth phase where
cells prepare for cell division in which the replicated DNA is equally divided between the
two daughter cells. The relative duration of these phases are usually cell type specific and
invariant for a certain cell type. The actual division of the genetic material and cell
division occur during mitosis or M phase. During this phase pr is dephosphorylated, by

as yet unidentified phosphatases to return the cells into the GI phase.

15

Estrogen and Cell Cycle regulation

With the identification of the events and the players controlling the cell division
cycle, the identity of the specific targets that estradiol regulates in order to induce
proliferation of breast tumor cells was addressed by a number of laboratories including
ours. The models to study breast cancer proliferation induced by estradiol were
developed in the 1970’s. These models consist of tumor derived cell lines from patients
with advanced breast cancer. One of the earliest successful attempts at this approach
resulted in the development of an estrogen receptor positive breast cancer cell line at the
Michigan Cancer Foundation by H.D. Soule, which was designated the MCF—7 cell line
(86, 87). This cell line grows in response to estrogen in tissue culture media, as well as
when implanted into nude mice. Fulthermore, tumors derived from MCF-7 cells in nude
mice are absolutely dependent upon estrogen for growth, as ovariectomized mice do not
form tumors without estrogen supplements (88). MCF-7 cells are very widely studied as a
model of estrogen dependent human breast cancers. Other human breast cancer cell lines
that are estrogen responsive include T47D, ZR-75-1 and CAMA-l (89-91). Studies using
these cell lines have identified targets of estrogen that are involved in proliferation, and
are building a comprehensive picture of how estrogens cause proliferation of breast
cancer cells. Treatment of estrogen responsive cells with estrogen enhances progression
of cells through the G1 phase of the cell cycle, and thereby increases the percentage of
cells that synthesize DNA. The duration of the other cell cycle phases is unaffected.

The protooncogenes c-fos, c-jun and c-myc are one of the first groups of genes
induced by estrogen (92). These protooncogenes play an essential role in cell cycle

progression. c-Myc is a transcription factor that is rapidly induced upon estrogen

16

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treatment of estrogen responsive cells (93). The c-Myc protein in turn induces
transcription of a number of cell cycle regulatory proteins including cyclin B, Cdc25A,
and decreases transcription of the p21wan kinase inhibitor in different cell types (94). c-
Myc’s essential role in estrogen-induced proliferation is highlighted by the fact that c-
myc antisense inhibits estrogen-induced proliferation (95). Furthermore, overexpression
of c-Myc can cause estrogen independent proliferation of MCF—7 cells (96). c-Jun
appears to play an important role in estrogen induced proliferation, as overexpression of
this protein in MCF-7 cells leads to proliferation in the absence of estrogen (97). c-Jun is
implicated in the regulation of cyclin D1 expression. Estrogen induced transcription of
cyclin D] has been shown to be regulated by a CRE element that binds to Jun-ATF-2
heterodimers (98). c-Fos also apparently plays an important role in estrogen-induced
proliferation since downregulation of c-Fos levels by an antisense oligonucleotide
strategy inhibits estrogen induced proliferation (99). Thus the protooncogenes regulated
by estrogen connect estrogen signaling to the cell cycle machinery, although the exact
mechanisms involved and targets in the cell cycle machinery of some of these immediate
early genes still remain to be determined.

The regulation of components of the cell cycle machinery by estrogen has been
extensively studied. Upon estrogen treatment of estrogen starved or antiestrogen treated
cells, an increase in Cdk4 and cyclin E-Cdk2 activities are observed and are accompanied
by increases in pr phosphorylation. An increase in the levels of cyclin D1 protein is one
of the earliest changes in the cell cycle regulatory machinery, occurring between 2-4
hours following estrogen treatment and has been proposed to account for the increase in

cyclin D1 associated Cdk4 activity. In MCF-7 cells the levels of cyclin B are constitutive

17

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and increases in cyclin E-Cdk2 activity cannot be explained by changes in cyclin E
levels. The increase in cyclin D1 has also been implicated in relieving inhibition of Cdk2-
cyclin B complexes by titrating the kinase inhibitor p21wan from the cyclin E-Cdk2
kinase complexes to cyclin Dl-Cdk4 complexes (100, 101). Because cyclin E-Cdk2
activity is essential for proliferation, it has been suggested that upregulation of cyclin D1
may thus mediate estrogen’s effect on cell cycle progression. This interpretation is
supported by the fact that overexpression of cyclin D1 can cause proliferation of MCF—7
cells arrested in G1 by antiestrogen treatment (96). The increase in proliferation upon
cyclin D1 overexpression is accompanied by an induction of Cdk4 and cyclin E-Cdk2
activities and pr phosphorylation and is consistent with increases in cyclin D1
mediating estrogen’s effects.

However, other studies have demonstrated a role for additional pathways in
mediating the proliferative effects of estrogens. c-Myc overexpression induces
proliferation in antiestrogen treated MCF-7 cells, and is accompanied by an increase in
cyclin E—Cdk2 activity and pr phosphorylation, but without an induction of cyclin D1
or Cdk4 activity (96). Thus, it appears that both cyclin D1 and c-Myc expression are
distinct targets and are independently required for the effects on estrogen on proliferation.
This model is supported by a recent study which showed that inhibition of Cdk4 activity
in MCF—7 cells, by infection with a p16mK4A encoding adenovirus, did not prevent the
induction of c-Myc or Cdc25A and downregulation of p21wafl by estrogen, but inhibited
cyclin E-Cdk2 activation, pr phosphorylation and proliferation (102). The fact that
estrogen could not induce proliferation in pl6mK4A overexpressing cells despite c-Myc

expression indicates the levels of c-Myc induced by estrogen are not sufficient to activate

18

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cyclin E-Cdk2 kinase activity and proliferation. It also suggests that estrogen is
regulating at least two distinct pathways affecting the cell cycle. In addition, a role for the

“’3“ and p27Kipl in mediating the proliferative effects

regulation of the Cdk inhibitors p21
of estrogen is suggested by the observations that both these proteins are downregulated
upon treatment of MCF-7 cells with estrogen (100, 101, 103). Furthermore, decreasing
the levels of these proteins using antisense oligonucleotides causes proliferation of MCF-
7 cells arrested in G1 by antiestrogen treatment (104). Significantly, a loss of p27”!
expression has been correlated with poor prognosis in breast cancer patients (105). The
loss of these kinase inhibitors would be expected to release the cyclin E-Cdk2 from
inhibition and thus induce proliferation. These studies bring into focus not only the
multiple targets that may be involved in estrogen mediated proliferation but also
mechanisms that may lead to cells becoming antiestrogen resistant.

Since the changes in cell cycle components induced by estrogen result in the
activation of Cdks, and pr inactivation may require two distinct Cdks (32, 106), a
question that is still unanswered is whether the ultimate effect of Cdk4 and Cdk2
activation by estrogen is to inactivate pr, or if there are additional targets of estrogen
that are required for its proliferative effects. The answer to this question is important not

only for understanding the pathways involved in estrogen mediated proliferation, but also

because of the implications this may have for acquired antiestrogen resistance.
Growth Factors and Breast Cancer

In addition to estrogens, a variety of growth factors including Insulin Like Growth

Factor-1 and 2 (IGF-1 and IGF-2), Epidermal Growth Factor (EGF), Transforming

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Growth Factor 01 (TGFa) and Fibroblast Growth Factor (FGF) have been identified as
potent mitogens for breast tumor proliferation (107-109). A number of studies have
suggested that estrogens affect breast tissue proliferation in an indirect manner by
inducing secretion of growth factors (110, 111). The secretion of both EGF and IGF-1
have been reported to increase upon estrogen treatment of breast cancer cell lines in vitro
(112, 113), and estrogen induced growth factors could partially replace estrogen to
promote tumor growth (114). A paracrine and/or autocrine role for growth factors in
mediating estrogen’s effects on mammary epithelial growth in vivo is supported by the
observation that proliferating breast epithelial cells in vivo are not ER positive (115). In
addition, breast epithelium from ER knockout mice transplanted into the fat pads of wild
type mice is capable of proliferation in response to estrogens. However, in the reverse
experiment, breast epithelial cells failed to proliferate, suggesting a role for paracrine
factors in the stroma, in the induction of breast tissue proliferation (116). However, the
role of paracrine factors induced by estrogens in breast cancer proliferation is still not
well defined. ER may exert a more direct effect on breast cancer proliferation since 70%
of breast cancers that are ER positive are responsive to the proliferative effects of
estrogen while ER negative tumors are rarely so. In addition, all estrogen responsive
breast cancer cell lines are ER positive and are responsive to estrogen in vitro in the
absence of stromal components. However, a role for autocrine factors secreted by
estrogen cannot be ruled out.

IGF-l is a potent mitogen for breast cancer cells (117). Alterations in the IGF-l
signaling pathway have been implicated in breast tumor progression (118), and have been

correlated with decreased disease free survival in ER positive breast cancer patients

20

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(119). The role of IGF-I in increasing breast cancer proliferation is not surprising since
IGF-l is required for the proliferation of normal breast tissue in viva. IGF-l knockout
mice fail to develop breast tissue beyond the prepubertal stage despite the presence of
estrogen, suggesting a critical role for IGF-l in breast tissue development and
proliferation (120).

IGF-l is a 70 amino acid protein secreted largely by the liver and the breast
stroma, (121) consistent with its postulated paracrine role in breast proliferation (122).
IGF-l binds to its cognate receptor, the IGF-l receptor (IGF-lR), which is a
heterotetrameric transmembrane protein tyrosine kinase expressed on the surface of
primary breast tumor cells (123). Upon activation by IGF-l binding, the IGF-IR
undergoes autophosphorylation on tyrosine residues. This stimulates the recruitment of
adapter proteins like Insulin receptor substrate-1 (IRS-l) (124), which in turn recruit and
activate signaling molecules resulting in the activation of the MAPK and PI3K signaling
pathway and increased proliferation (125, 126). Interestingly, IGF-l induced proliferation
is inhibited by antiestrogen treatment in vitra, suggesting that these two signaling
pathways interact (127, 128). A number of studies have reported regulation of
intermediates in the IGF-l signaling pathway, including the levels of IGF-IR and IRS-1
protein and the phosphorylation of IRS-1, PI3K and AKT, the downstream signaling
molecules in the IGF-l signaling pathway by estrogens and antiestrogens. These data
indicate an indirect effect of estrogens on proliferation (129-133). Similarly, IGF-l
activates the MAPK signaling cascade in MCF—7 cells, and estrogens have been reported
to regulate the MAPK signaling pathway (134, 135), although there are some reports to

the contrary. Despite the above mentioned data, it is still unclear if antiestrogens inhibit

21

growth factor induced proliferation by downregulating components of growth factor
signaling, or if both growth factor and ER signaling are independently required for
proliferation. In the former case an upregulation of growth factor signaling components

could result in antiestrogen resistance, whereas in the latter case this would not be true.

lGFl
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PKB(AKT)*

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Cyclin D1 IRS], 1G " R

Figure 1.3. Interaction of IGF-I and estrogen signaling. IGF—l signaling and signaling
via estrogen receptor can potentially interact at multiple levels to regulate the
proliferation of breast cancer cells. IGF-l binds to its membrane bound receptor (IGF-
1R) and leads to autophosphorylation of the receptor. Phosphorylated IGF-lR then
recruits IRS-1, which causes membrane localization of PI3K, a lipid inositol kinase. This
further recruits enzymes that lead to a cascade of signaling events including AKT
activation. Activated AKT causes increased cyclin D1 stability and is one of the ways
that IGF-l signaling upregulates cell cycle components. Estrogen by activating the
estrogen receptor can also regulate the transcription of cyclin D1. Thus, both IGF-I and
estrogen could be independently regulating the same cell cycle components However,
both IGF-lR and IRS-l genes are transcriptionally upregulated by estrogen, and an
increase in IGF-l signaling could explain estrogen’s proliferative effects on breast cancer
cells.

22

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Antiestrogen therapy and antiestrogen resistance

Once the role of estrogen in breast cancer proliferation was recognized, efforts to
develop a rational therapy that would antagonize the effects of estrogen led to the
development of antiestrogenic compounds. Tamoxifen is a nonsteroidal antiestrogen and
is the most widely used therapy to treat estrogen responsive breast tumors. Later research
led to the development of steroidal antagonists of estradiol, such as ICI 182,780 (136).

Tamoxifen is a partial estrogen antagonist. In certain tissues it acts as an agonist
and activates estrogen responsive genes, while in others it antagonizes estrogen’s effects.
Tamoxifen acts as an antagonist in breast tissue, while it is an agonist in some other
tissues such as the uterine endometrium (137), cardiovascular and skeletal tissues. In the
latter two tissues, the effects of estrogens are thought to be protective against
cardiovascular disease and osteoporosis (138, 139). The protective effects of estrogen on
the above tissues are not inhibited by Tamoxifen, making it a specific treatment for breast
cancer with minimal adverse effects. Although both steroidal and nonsteroidal
compounds are competitive inhibitors of estrogen binding to the ER, they have somewhat
different actions. Tamoxifen has a 100 fold lower binding affinity to the estrogen receptor
compared to estradiol and the other steroidal antagonists such as ICI 182,780. Binding of
Tamoxifen to the ligand binding domain of estrogen receptor does not affect dimerization
or DNA binding of ER but causes changes in ER conformation (22) that result in
recruitment of corepressors instead of coactivators to the promoter (26), and concurrent
inhibition of transcription of genes important for proliferation. Interestingly, recruitment
of a coactivator to the Tamoxifen bound ER, by substituting its nuclear receptor binding

domain with that of a corepressor, induces transcription from target genes by Tamoxifen

23

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bound ER (26). The partial agonist activity of Tamoxifen in some tissues is yet to be
explained, but may be due to differential expression or binding of
coactivator/corepressors in different tissues. In contrast, the steroidal antagonists such as
ICI 182,780 have no agonist effects, in part explained by the degradation of the ER
induced by these compounds and leads to adverse effects in other tissues where estrogen
is protective.

Numerous studies have shown that cells that are initially sensitive to Tamoxifen
eventually stop responding to Tamoxifen over the course of therapy (140). This acquired
resistance is recapitulated in both in viva and in vitra models using MCF-7 cells. Work by
K. Osborne has shown that MCF—7 cells implanted in nude mice initially do not grow in
the presence of Tamoxifen (141). However, over the course of 3-4 months, these tumors
develop to a state of antiestrogen resistance (142). Some of these Tamoxifen resistant
tumors are responsive to ICI 182,780, suggesting that the ER is still required for their
proliferation (143). However, the adverse effects of steroidal antagonists, such as
exacerbated bone loss, limit the use of these drugs as second lines of treatment. There is
still limited clinical information on the development of resistance to steroidal antagonists
such as ICI 182,780, because this compound has been used in clinical trials only recently
(143). However, resistance to ICI 182,780 can potentially occur, as some Tamoxifen
resistant tumors do not respond to steroidal antiestrogens (144) and ICI resistant clones
can be selected in vitra (145) and in mice (146) and this can occur without a loss of ER
(147-149).

Because antiestrogen resistance is a major cause of treatment failure and mortality

in breast cancer, understanding the mechanisms of this resistance is critical to the

24

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development of improved therapeutics for these patients. It is apparent that breast cancers
can employ a variety of mechanisms to acquire antiestrogen resistance (150). Altered
metabolism of antiestrogens by tumor cells could potentially contribute to antiestrogen
resistance, as could increased production of estrogen by patients undergoing prolonged
therapy with Tamoxifen (13). The loss of estrogen receptor (ER) would result in
antiestrogen resistance, with a subset of tumors showing receptor loss, though most
antiestrogen resistant tumors retain ER expression (151). Expression of variant forms of
estrogen receptor may be involved in the development of antiestrogen resistance, but the
evidence for such a mechanism is still not definitive (152). Additionally, changes in the
expression of coactivators/corepressors are another potential mechanism for the
generation of resistance. Changes in growth factor signaling have also been reported to
contribute to antiestrogen resistance. Epidermal growth factor activates the EGF-
Receptor (EGF-R), a membrane bound tyrosine kinase receptor that leads to proliferation
in estrogen dependent and independent breast cancer cells and of normal breast epithelial
cells (114, 153). Expression of the EGF-R has been correlated with Tamoxifen resistance
(154),(155) and the related receptor erbBZ has been shown to decrease the requirement
for estrogen for growth (156). Also, changes in the expression of EGF/HER2 ligands that
activate the EGF-R have been correlated with antiestrogen resistance (157). Similarly,
studies have suggested that overexpression of components of the IGF-l signaling
pathways may contribute to a loss of estrogen dependence for growth. MCF—7 cell lines
that overexpress IGF-l receptor (IGF-lR) or the IGF-IR downstream target IRS-1, have
reduced requirements for estrogen for proliferation. Also cells expressing FGFOt have

reduced requirements for estrogens for proliferation, and are able to proliferate in the

25

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presence of Tamoxifen and ICI 182,780 (144, 158-160). Similarly, MCF—7 cells
overexpressing TGFor show an estrogen independent and Tamoxifen resistant phenotype
(161). In summary, a variety of events, including a loss of ER, altered metabolism of
Tamoxifen and upregulation of growth factor signaling pathways can lead to the
development of antiestrogen resistance.

Alterations in the cell cycle components regulated by estrogen could also
potentially lead to the development of antiestrogen resistance. Studies using in vitra
models have suggested that alterations in a number of cell cycle components including
overexpression of c-Myc, c-Jun, cyclin D1 and loss of the negative regulators of cell
cycle p21wan and p27Kipl can result in antiestrogen resistance (96, 97, 104). Though
overexpression of cyclin D1 and c-myc as well as loss of p27Kipl has been documented in
a fair percentage of breast cancers, the clinical relevance of these findings at present is
not fully defined, nor are the levels of these proteins used for diagnostic or therapeutic
decisions at present.

The goal of this study was to define the events in the cell cycle that are regulated
by estrogens and whose deregulation may lead to the development of antiestrogen
resistance. In particular, we have focused on studying the effects of pr inactivation on
antiestrogen resistance, since estrogen appears to mediate its proliferative effect by
causing pr inactivation and alterations in the pr pathway have been reported in a
significant percentage of breast cancer patients. In addition, the effects of activating the
IGF-l signaling pathway on antiestrogen resistance were assessed, as numerous studies

have suggested that estrogen regulates breast cancer proliferation indirectly by regulating

IGF-l signaling. Thus activation of IGF-I signaling could potentially lead to antiestrogen

26

resistance. We therefore tested the hypothesis that antiestrogens downregulated IGF-l
induced signaling events in MCF-7 cells and further if activation of IGF-1 signaling

could confer antiestrogen resistance.

27

.IJ

II

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Love, R. R., Wiebe, D. A., Newcomb, P. A., Cameron, L., Leventhal, H., Jordan,
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Wiebe, V. J ., Osborne, C. K., Fuqua, S. A., and DeGregorio, M. W. Tamoxifen
resistance in breast cancer, Crit Rev Oncol Hematol. 14: 173-88., 1993.

Osborne, C. K., Hobbs, K., and Clark, G. M. Effect of estrogens and antiestrogens
on growth of human breast cancer cells in athymic nude mice, Cancer Res. 45:
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Osborne, C. K., Coronado, E. B., and Robinson, J. P. Human breast cancer in the
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Lykkesfeldt, A. E., Larsen, S. S., and Briand, P. Human breast cancer cell lines
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Arteaga, C. L., Carty-Dugger, T., Moses, H. L., Hurd, S. D., and Pietenpol, J. A.
Transforming growth factor beta 1 can induce estrogen-independent
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4: 193-201., 1993.

50

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CHAPTER 2

Reversal of an Antiestrogen-Mediated Cell Cycle Arrest of MCF-7 Cells by Viral

Tumor Antigens Requires the Retinoblastoma Protein-Binding Domain

ABSTRACT

Proliferation of MCF—7 cells is estrogen dependent and antiestrogen sensitive. In the
absence of estrogens or presence of antiestrogens MCF-7 cells arrest in the G1 phase of
the cell cycle, and this arrest is associated with an accumulation of the active,
hypophosphorylated form of the retinoblastoma protein (pr). Because active pr
negatively regulates passage from G1 to S phase, this suggests that pr is a crucial target
of estrogen action, and that its inactivation might lead to antiestrogen resistance. We
tested this hypothesis by expressing viral tumor antigens (T antigens), which bind and
inactivate pr, in MCF—7 cells, and determining the effects on cell proliferation in the
presence of antiestrogens. The results of these experiments demonstrate that T antigen
expression confers antiestrogen resistance to MCF-7 cells. Using a panel of mutant T
antigens, we further demonstrate that the pr-binding, but not the p53 binding domain is
required to confer antiestrogen resistance. Thus, pr is an important target of estrogen

action, and its inactivation can contribute to the development of antiestrogen resistance.

 

Note. The content of this chapter has been published as Varma, H., and Conrad, SE.
2000. 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.

51

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INTRODUCTION

Approximately 40% of human breast cancers require estrogens such as 1713-
estradiol for growth (1). In the absence of estrogens or the presence of antiestrogens,
proliferation of these tumor cells is inhibited and they accumulate in the G1 phase of the
cell cycle (2, 3). Because of their ability to inhibit proliferation, antiestrogens are often
used to treat breast cancer patients. However, a significant proportion of tumors that
initially respond to antiestrogen therapy eventually progress to a resistant state in which
cell proliferation is no longer dependent on estrogens or inhibited by antiestrogens.
Understanding the molecular mechanisms by which estrogens and antiestrogens regulate
passage from G1 to S phase, and those by which cells become antiestrogen resistant, are
therefore important goals that may lead to improvements in breast cancer diagnosis and
treatment.

Transit through G1 into S phase is regulated by cyclin dependent kinases (Cdks),
a family of protein kinases that are activated by association with cyclins. The Cdks that
regulate passage through G1 include Cdk4 and Cdk6, which are activated by the D-type
cyclins, and Cdk2, which is activated by both cyclin E and cyclin A (4, 5). One important
target of G1 cyclin/Cdk complexes is the tumor suppressor protein pr (6). In its
hypophosphorylated form, pr associates with members of the E2F family of
transcription factors, and prevents transcription of genes required for entry into S phase.
Upon phosphorylation of pr, E2F transcription factors are released and activate target
genes that promote S phase entry (Figure 2.1A) (7, 8).

The effects of estrogen and antiestrogen treatments on cell cycle progression have
primarily been investigated in the MCF—7 cell line, which is an estrogen receptor positive
human breast cancer cell line. Proliferation of MCF-7 cells is estrogen-dependent and

antiestrogen sensitive both in vitro and in vivo (2, 9, 10), and in the presence of

52

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antiestrogens these cells accumulate in G1. Estrogen treatment of G1 -arrested MCF-7
cells induces S phase entry, and this induction is associated with increases in cyclin D1
protein levels, cyclin D1/Cdk4 kinase activity, Cdk2 kinase activity, and pr
phosphorylation (ll-l4). In addition, overexpression of cyclin D1 in the presence of
antiestrogens leads to pr phosphorylation, and is sufficient to promote proliferation for
at least one cell cycle (15, 16).

One explanation for the findings outlined above is that pr is the ultimate target
of the proliferative effects of estrogen, and that overexpression of cyclin D1 promotes
proliferation in the presence of antiestrogens by increasing pr phosphorylation via the
activation of cyclinDl-Cdk4 complexes. However, it is also possible that functions of
cyclin D1 other than activating Cdk4 contribute to or are responsible for antiestrogen
resistance. For example, regulation of cyclin E-Cdk2 kinase activity by estrogen and
antiestrogens occurs in the absence of changes in cyclin B or Cdk2 protein levels. To
account for this observation, it has been proposed that induced or overexpressed cyclin
D1 protein binds and sequesters the Cdk inhibitor p21wafl, leading to activation of cyclin
E-Cdk2 complexes (12, 13). Another potential mechanism of cyclin D1 action is via its
interactions with cellular transcription factors. Cyclin D1 binds to and modulates the
activity of several transcription factors, including the estrogen receptor (17, 18) and a
myb-like transcription factor DMPl (19, 20), and either of these activities could have
profound effects on cell proliferation.

If pr is the ultimate target of the proliferative effects of estrogen, one would
predict that inactivation of this tumor suppressor protein would be sufficient to confer
antiestrogen resistance to MCF-7 cells. Alternatively, if functions of cyclin D1 other than
promoting pr phosphorylation are critical for its effects on estrogen dependence, then
inactivation of pr would not be sufficient to promote antiestrogen resistance. To

directly test the effect of pr inactivation on antiestrogen sensitivity, we have expressed

53

 

Fig

Estrogen
V P
Cyclin/CDK _,,---
w 1’ pRB P
"7333.5“ , _ p E2F Active
E2F " KEZF SPhase Entry
B
r (31.1" : 988:}
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. ' y, ._ p azr Active
1221“ .‘ E2F . SPhase Entry

Figure 2.1. Model for pr inactivation by (A) estrogen treatment and (B) LT expression
in MCF—7 cells.

54

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the simian virus 40 (SV) or polyoma virus (Py) large T antigens (LTs) in MCF—7 cells.
Both SVLT and PyLT bind to and inactivate pr (21-24), thereby allowing E2F to
activate transcription and promote S phase entry (Figure 2.1B). SVLT has several other
activities in addition to pr-binding, including the ability to inactivate the tumor
suppressor protein p53 (25, 26). PyLT shares most of these properties, but differs from
SVLT in that it does not bind to p53 (21, 27). Using wild type and mutant forms of the
viral proteins, we demonstrate that expression of either SV40 or polyoma virus LT is
sufficient to confer antiestrogen resistance to MCF-7 cells. This effect is dependent upon
an intact pr binding domain, but does not require a p53 binding domain. These data
establish that pr or a related protein is an important functional target of estrogen, and
suggest that inactivation of normal pr function could play a role in the development of

antiestrogen resistance during tumor progression in vivo.

MATERIALS AND METHODS

Cell Culture. MCF-7 cells were obtained from Dr. Michael Johnson (Lombardi Cancer
Center) and were maintained in IMEM media (Biofluids Inc., Rockville, MD)
supplemented with 5% fetal bovine serum (Hyclone), penicillin (100 units/ml) and
streptomycin (100 ug/ml). To study the effects of estrogen and antiestrogens, cells were
cultured in phenol red free IMEM supplemented with 5% charcoal stripped fetal bovine
serum (Hyclone), with or without the addition of 0.2 nM 17B-estradiol (Sigma), 1 M 4-
hydroxytamoxifen (Sigma), or 100 nM ICI 182,780 (Zeneca Pharmaceuticals)

Antibodies and Plasmids. The antibody to SVLT was a supernatant of the L19

55

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hybridoma cell line, which was a gift of Dr. Ed Harlow (28). A rat monoclonal antibody,
Ab-l (Oncogene Science) was used to detect PyLT by indirect immunofluorescence. A
polyclonal rat antiserum was used for PyLT immunoblotting, and was a gift of Dr.
Michele Fluck. Antibodies for pr, cyclin A, and p53 were from Pharmingen. The
mouse a-BrdU monoclonal antibody was obtained from Boehringer Mannheim, and
sheep or—BrdU was from Fitzgerald Industries, Concord, MA. FITC conjugated goat or—
rat, donkey or-sheep, and goat a-mouse antibodies were obtained from Sigma
Biochemicals. Rhodamine Red-X conjugated donkey or-mouse antibody was from
Jackson Immunoresearch.

The plasmids encoding wild type SVLT, PVUl, and 2809 were obtained from Dr.
Charles Cole (29, 30). The SV40 genome in all of these plasmids contains a 6 bp deletion
at the viral origin of replication that renders them replication incompetent (30-32). The
truncation mutant (Tl-259) was provided by Dr. Ellen Fanning (33). The T antigen
encoded by this mutant lacks helicase and ATPase activity and will therefore not promote
replication from the viral origin (34-36). The plasmid pCMVLT, which contains a cDN A
encoding PyLT, was obtained from Dr. Brian Schaffhausen (37). The pneoLTl and
d1141-encoding vectors used for stable transfections were provided by Dr. Michael Bastin

(23, 38). The p53 dominant negative mutant (de1239) was a gift of Dr. John Minna (39).

Transfections. For transient transfections, 5x105 MCF-7 cells were plated in 60 mm
tissue culture dishes containing 2-18 mm cover slips and incubated at 37°C overnight.
Plasmid DNA was added to the cells using the Superfect reagent (Qiagen) following the
protocol suggested by the manufacturer. SV40 LT transfections were done using 2.5 and
5 ug of DNA per dish, and pCMV-LT transfections were done using 0.3 pg DNA per
dish. These amounts were chosen because they gave the highest transfection efficiencies
in pilot experiments. After 3 hr, the transfection medium was removed and replaced with

phenol red free IMEM supplemented with 5% charcoal stripped serum and 100 nM ICI

56

 

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182,780. Cells were incubated for 43 hr and then labeled with 25 M 5-bromo-2'—
deoxyuridine (BrdU) for 5 hr. For control proliferating cultures, cells were grown in
medium containing charcoal stripped serum supplemented with 0.2 nM 17B-estradiol.
For stable transfections, 1 pg of DNA (pneoLTl or dll4l) was introduced into MCF—7
cells using Lipofectin reagent (Gibco BRL). Colonies were selected for G418 resistance
in medium containing 400 ug/ml G418 and expanded into clonal cell lines. PyLT

expression in these cell lines was assayed by Western blotting.

Indirect Immunofluorescence. Forty-eight hours after transfection, cells on coverslips
were fixed with ice cold acetone/methanol (50:50v/v), and washed with phosphate
buffered saline (PBS). The following procedures were used for antibody staining, with
PBS washes (3X) being conducted between each treatment. Coverslips containing fixed
cells were incubated with a primary antibody against SVLT or PyLT for 45 min at 37°C,
then with the appropriate fluorochrome-conjugated secondary antibody for 1 hr at room
temperature. Following this procedure, cells were re-fixed in a 50mM glycine, 50%
ethanol solution (pH 2.1) for 45 min at room temperature. Samples were then treated with
4 N HCl for 15 min to denature the DNA. The acid was aspirated, and coverslips were
washed in PBS to neutral pH (7.0). Samples were then incubated with an appropriate
anti-BrdU antibody (sheep for SVLT and mouse for PyLT) for 45 min at 37°C, washed,
and incubated with a second fluorescent antibody (donkey anti-sheep FITC for SVLT and
donkey anti-mouse rhodamine Red X for PyLT). Finally, the coverslips were extensively
washed with PBS, mounted on glass slides, and viewed under a flourescence microscope
(Olympus). The percentage of cells that were positive for LT (or p53) alone, BrdU alone,
or both, were counted in each transfection. More than 100 LT-positive and 100 LT-
negative cells were counted in each experiment. The images shown in Figure 2.3 were
taken on a Meridian Instruments confocal microscope, and processed with Insight IQ

computer software to generate single color and overlay images.

57

 

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RESULTS

Inhibition of DNA synthesis by antiestrogens in MCF-7 cells. Antiestrogens inhibit
proliferation of MCF—7 cells. This is demonstrated in Figure 2.2A, in which the
percentages of cells incorporating BrdU into DNA are compared in cultures incubated in
basal medium containing 5% charcoal stripped serum (CSS), and in this medium
supplemented with 17B-estradiol (E), 4-hydroxytamoxifen (TAM) or ICI 182,780 (ICI).
As previously reported by others, the dependence of MCF—7 cell proliferation on
exogenous E in cell culture is not absolute (2, 40, 41). E stimulated proliferation, but
there was considerable BrdU incorporation in the basal medium in these assays. TAM is
a non-steroidal antiestrogen with both agonist and antagonist activities. It acted as an
antagonist in these assays, and decreased proliferation below the level observed in CSS
alone. ICI is a steroidal antiestrogen with no agonist activity. As previously reported, it
was a more potent inhibitor of MCF-7 cell proliferation than TAM (41), and decreased
the percentage of cells incorporating BrdU to approximately 3-5%. In addition, MCF—7
cell derivatives that are resistant to ICI are cross-resistant to TAM, while TAMr cells are
often sensitive to ICI (42, 43). The ability of a treatment to confer ICI resistance is
therefore likely to also confer TAM resistance, while the converse is not true. For these

reasons, ICI was the antiestrogen used in the remainder of our experiments.

Expression of SV40 large T antigen confers antiestrogen resistance to MCF-7 cells,
and the pr binding function is required for this effect. To determine the effect of
SVLT expression on antiestrogen sensitivity, plasmids encoding either wild type or
mutant SVLT proteins were introduced into MCF—7 cells, and the ability of the
transfected cells to incorporate BrdU into DNA in the presence of ICI was determined as

described in Materials and Methods.

58

Table 2.1. Characteristics of wild type and mutant viral T-antigens used

 

 

in study.
pr p53

LT Protein binding . binding Mutation Reference
PVUl (SV) - + delaa-107/112 (56)
2809 (SV) + - ins 409 (4aa) (29)

T l -259 (SV) + - truncation (33)

PyLT + - none (23, 37)

d1141 (Py) - - del aa 141/146 (38)

 

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SVLT is a multifunctional protein that interacts with a number of cellular proteins,
including the pr family of proteins (pr, p107, p130), p53, p300 and TBP (21, 27). The
mutants used in these experiments (Table 2.1) were defective in one or more of these
activities that seemed likely to play a role in promoting antiestrogen resistance. As
described in Materials and Methods, none of the constructs encoding SVLT were capable
of replicating extrachromosomally, and any BrdU incorporation detected therefore
represented cellular DNA replication.

Representative micrographs of cells expressing SVLT and/or incorporating BrUrd
are shown in Figure 2.3. The percentages of SVLT positive and SVLT negative cells in
each transfected culture that were also BrdU positive was determined, and the results of
this analysis are shown in Figure 2.2B. The percentage of SVLT negative cells that
incorporated BrdU in the transfected cultures (3-5%) was similar to untransfected, ICI
treated controls (Figures 2.2A and 2.2B), indicating that the transfection protocol did not
cause proliferation. In contrast, cells expressing wild type SVLT incorporated BrdU at a
level comparable to the cycling (E) population. These results indicate that SVLT is able
to completely overcome an ICI-mediated cell cycle arrest in this assay.

The PVUI mutant is defective in binding to pr family members. In the presence
of ICI, cells expressing PVUl protein incorporated BrdU to the same low extent as
untransfected cells (Figures 2.2B and 2.3). Both the transfection efficiency of the PVUl
construct, and the level of expression of PVUl protein in transfected cells were
equivalent to the wild type (Figure 2C), indicating that the defect in this mutant was not
due to a failure of the protein to be expressed. We therefore conclude that the pr-
binding domain of SVLT is absolutely required to overcome an ICI induced cell cycle
arrest.

Two SVLT mutants defective in p53 binding were also examined. One (2809)

contains an insertion of 4 amino acids at position aa409, and the second (Tl-259) is a

60

 

Figure 2.2. Ability of wild type and mutant LT proteins to promote antiestrogen
resistance. MCF—7 cells were transfected with LT-encoding plasmids, labeled with BrdU,
and stained for LT expression and BrdU incorporation as described in Materials and
Methods. (A) The percentage of cells in untransfected cultures that incorporated BrdUrd
during a 5 hr labeling in the presence of CSS, CSS+E, CSS+TAM or CSS+ ICI. The
results represent the mean +/- SE of an experiment done in triplicate. (B) The percentages
of LT positive (filled bars) and LT negative (open bars) cells that also incorporated BrdU
in the presence of ICI in transfected cultures. The percentage of cells that incorporated
BrdU during a 5 hr labeling in the presence of E or ICI in untransfected cultures are
shown on the left side of the figure The results represent the mean 3; SE of at least three
independent experiments. (C) A representative western blot showing expression levels of
wild type and mutant SVLTs. Arrows point to the T1-259 truncation mutant, and to a
smaller (20—25 kDa) form of LT detected in cells transfected with the 2809 mutant.
Values for the transfection efficiency (percentage) of each construct are represented as
the mean i SE for at least three independent experiments.

61

% Brdl Erd Positive Cells

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62

Overlay

      

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2809

PyLT

Figure 2.3. Double indirect immunofluorescence of transfected cells. MCF—7 cells were
transfected with wild type or mutant LT-encoding plasmids, incubated in medium
containing ICI, and labeled with BrdU as described in Materials and Methods. SVLT was
detected using a rhodamine-conjugated secondary antibody (red fluorescence), and BrdU
incorporation with an FITC-conjugated secondary antibody (green fluoresence). PyLT
was detected using an FITC-conjugated secondary antibody, and in this case BrdU
incorporation was detected with a rhodamine-conjugated secondary antibody. Overlay
images were generated to show nuclei that were positive for both LT and BrdU, which
appear yellow in the photograph.

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truncation mutant encoding the first 259 amino acids of the protein. Both the 2809 and
Tl-259 mutants were 60-70% as effective as wild type SVLT at inducing proliferation in
the presence of ICI (Figure 2.2B). To investigate potential reasons for this small, but
reproducible decrease in the ability of these mutants to overcome an ICI-induced arrest,
both the transfection efficiency of the DNA constructs and the level of expression of the
encoded proteins in transfected cell populations were examined. As shown in Figure
2.2C, the percentage of cells expressing detectable SVLT in 2809 and T1-259 transfected
cultures was approximately 2-fold lower than with the wild type, although the intensity of
staining in LT positive cells was not detectably different between the three constructs. In
agreement with these observations, overall T1-259 protein levels in transfected cultures
were approximately 2-fold lower than wild type as determined by western blots.
Interestingly, cultures containing the 2809 mutant expressed very low levels of full length
LT protein, although they incorporated BrdU with approximately the same efficiency as
Tl-259. However, it was noted that 2809-transfected cells expressed relatively high
levels (equal to that of the T1-259 mutant) of a small protein of approximately 20-25
kDa. Since the antibody used in these western blots recognizes an N-terminal epitope of
SV- LT (28), the 20-25 kDa peptide may be an N-terminal fragment that retains the pr
binding domain and has activity in proliferation assays. Alternatively, all of the
proliferative activity of the 2809 mutant may be due to the small amount of full-length
protein present in the transfected cells. In summary, two different SVLT mutants that are
defective for p53 binding were able to induce proliferation in ICI treated MCF—7 cells.
However, the magnitude of the effect was significantly less than that seen with the wild
type protein. Due to differences in the levels of expression of the encoded proteins, the
data obtained did not allow us to distinguish whether the decreased ability of the 2809
and T1-259 mutants to overcome an antiestrogen-induced arrest was due to decreased

expression of these proteins, or if they were truly less active than wild type SVLT.

lnact
possfl
renst
hhif
enter
ras

mien
lfigU'
than

them

rned

SEE
the
the

ICE

Inactivation of p53 does not confer antiestrogen resistance. To further investigate the
possibility that p53 inactivation played a role in SVLT’s ability to confer antiestrogen
resistance, we examined the ability of PyLT to promote proliferation of ICI-arrested
MCF—7 cells. PyLT binds to pr, but does not bind or inactivate p53 (21, 27). A plasmid
encoding wild type PyLT (pCMVLT) was transfected into MCF—7 cells, and proliferation
was assayed in the presence of ICI as described above. Representative fluorescence
micrographs are shown in Figure 2.3, and summaries of the experiments are shown in
Figures 2.2B and 2.4. Although the average induction of proliferation was slightly lower
than seen with SVLT, the difference was not statistically significant. These results
therefore indicate that PyLT is as efficient as SVLT in overcoming an antiestrogen-
mediated arrest of MCF—7 cells, and establish that p53 inactivation is not required for this
effect.

The results presented above indicated that p53 inactivation was not required to
confer antiestrogen resistance to cells under conditions where pr family members were
inactivated. To examine the effects of p53 inactivation alone on antiestrogen sensitivity, a
plasmid encoding de1239, a dominant negative (dn) p53 mutant (44), was transfected into
MCF—7 cells. Expression of this protein did not increase BrdU incorporation above that
seen in untransfected cells (Figure 2.4). In addition, de1239 expression did not enhance
the level of proliferation induced by PyLT alone in co-transfection experiments. We
therefore conclude that p53 inactivation alone is not sufficient to confer antiestrogen
resistance, and that it does not enhance the resistance conferred by pr inactivation, in

MCF-7 cells.

PyLT confers antiestrogen resistance in long term assays. It was previously reported
that over-expression of cyclin D1 conferred antiestrogen resistance to MCF-7 cells in
short term assays, but was not sufficient to promote long term growth in the presence of

antiestrogen (15). We therefore wanted to determine if LT expression was sufficient to

65

Fig
MC
(del
del:
EXP
CFC]
inde

U iitransicctcd

 

W
7’ 35 ‘ .. .
U ' I lranslcctcd
3’ 30
.r.
E": 25
a. 20 ,
g 15 -
'2 10 j
a:
$3 5"
0 L .. - -_- ...... .
15 PyL'l‘ del239 P\ 1.1
id61239
+1Cl

Figure 2.4. Effects of PyLT and dominant negative p53 expression on ICI sensitivity in
MCF—7 cells. MCF7 cells were transfected with plasmids encoding PyLT, a dn p53
(de1239), or both. BrdU incorporation in the presence of ICI was determined in PyLT or
del239 positive cells (filled bars) and PyLT or del239 negative cells (open bars), and is
expressed as a percentage of the total cells. BrdU incorporation was also measured in
cycling (E) cells as a positive control. Results represent the mean +/- SE of three
independent experiments.

66

Fig]
MC
Sele
ass;

of f
116g
hip
CO“
“er
blOl

A
WTl WT4 WT5 d114l

. ms

 

11
c
:5

23?! m gaff

 

WTI WT4 WT5 dll4l
ICI - + .. + _ + _ _:

 

Figure 2.5. Effects of PyLT expression on ICI sensitivity in stably transfected cells.
MCF—7 cells were transfected with vectors encoding PyLT, and G418r colonies were
selected and propagated as described in Materials and Methods. A) G418r cell lines were
assayed for PyLT expression and pr phosphorylation by western blotting. Cell lines
WTl and WT5 expressed wild type PyLT. dl-141 cells expressed a pr binding mutant
of PyLT. WT4 was a G418r clone that did not express detectable PyLT, and served as a
negative control. Hyperphosphorylated pr (ppr) migrates more slowly than the
hypophosphorylated form (pr) on these gels. B) Cell lines were plated in medium
containing 5% FBS, treated with either ICI (+) or vehicle (-) for 48 hrs, and cell extracts
were prepared and analyzed for pr phosphorylation and cyclin A expression by western
blotting. The blots were also probed for actin as a control for protein loading.

67

cont
Con
intn
sele
into
line
bin.
line
con
sirr

(Fl;

fit";
We
she

tre

“’1
the
sir

{Cc

confer antiestrogen resistance to MCF—7 cells in long term proliferation assays.
Constructs encoding either a wild type or pr binding mutant (d1141) of PyLT were
introduced into MCF-7 cells using the pneo-LTl vector, which contains a G418r gene for
selection of stably transfected cells. Individual G418r colonies were selected, expanded
into cell lines, and tested for expression of PyLT protein by western blotting. Two cell
lines expressing the wild type protein (WT 1 and WT5) and one expressing the pr
binding mutant (dl 141) were chosen for further analysis (Figure 2.5 A). One G418r cell
line that did not express detectable levels of PyLT protein (WT4), was used as a negative
control. All of these cell lines grew well in the presence of 5% FBS, and contained
similar amounts and ratios of hyper- and hypo-phosphorylated pr in this medium

(Figure 2.5 A).

The effects of ICI treatment on proliferation of the PyLT expressing cells were
evaluated in several ways. In Figure 2.5B, pr phosphorylation and cyclin A expression
were examined by western blotting after 2 days of ICI treatment. Previous studies have
shown that both pr phosphorylation and cyclin A expression are inhibited by ICI
treatment of the parental MCF-7 cell line (14). In agreement with this,
hyperphosphorylated pr and cyclin A protein were undetectable in ICI-treated WT4
control cells, which do not express PyLT. In contrast, WTl and WT5 cells, which express
wild type PyLT, contained both hyperphosphorylated pr and cyclin A when grown in
the presence of ICI. Cells expressing d1141, the pr binding mutant of PyLT, had a
similar phenotype to WT4 cells, confirming that the pr-binding domain is absolutely

required for viral tumor antigens to promote proliferation in the presence of ICI. The

68

.~

eltecl
by c
incor
IllOSr

resis

phc
inm
test

the

ant
\‘ii
Ti

its

at

effects of ICI treatment on pr phosphorylation and cyclin A expression were paralleled
by changes _ in cell proliferation, as measured by either 3H-thymidine or BrdU
incorporation (data not shown). Taken together, the results in stable transfectants confirm
those seen in transient assays, and indicate that expression of PyLT confers antiestrogen

resistance to MCF-7 cells

DISCUSSION

Previous evidence linking estrogen and antiestrogen treatments to pr
phosphorylation suggested that pr might be a crucial target of estrogen, and that
inactivation of this tumor suppressor protein might lead to antiestrogen resistance. We
tested this possibility by expressing viral T antigens in MCF-7 cells, and determining
their effects on antiestrogen sensitivity. Expression of either the SV40 or polyoma virus
large T antigen promotes MCF—7 cell proliferation in the presence of the pure
antiestrogen ICI 182,780. Furthermore, this effect is dependent upon the ability of the
viral proteins to bind and inactivate pr, but is independent of their ability to bind p53.
Thus, pr is an important target of estrogens in regulating MCF—7 cell proliferation, and
its inactivation can contribute to the development of antiestrogen resistance.

The fact that both pr inactivation and overexpression of cyclin D1 (15, 16)
promote MCF—7 cell proliferation in the presence of antiestrogens confirms an important
role for the cyclin D/pr pathway in estrogen dependence. It also suggests that the
primary mechanism by which cyclin D1 overcomes an antiestrogen-mediated arrest is by

activating Cdk 4/6, which in turn phosphorylates pr. As discussed previously, Cdk2

69

acti‘

S€C(

thei

01

[Ill

3C1

lei

sir

be

activity is inhibited in antiestrogen treated MCF—7 cells, and it has been proposed that a

second role for cyclin D1 in promoting antiestrogen resistance is to sequester p21wa”,

thereby leading to Cdk2 activation. The results reported here suggest that either

antiestrogen treatment fails to inhibit Cdk2 activity in MCF—7 cells expressing T antigens,

or that the requirement for Cdk2 activity is bypassed in these cells. Since p21wan

transcription is induced by p53 (45), one mechanism by which SVLT could lead to Cdk2

activation would be by binding and inactivating p53, leading to decreases in p21wan

levels. However, this does not appear to play a role in promoting antiestrogen resistance,
since LTs that do not inactivate p53 induce proliferation in the presence of ICI, and since
a dn p53 mutant does not induce proliferation in the presence of ICI. The mechanisms by
which LTs overcome the inhibition of Cdk2 activity by antiestrogens therefore remain to

be elucidated.

Although our results clearly demonstrate that an intact pr -binding domain is
required, they do not establish if pr inactivation alone is sufficient to confer
antiestrogen resistance to MCF—7 cells. The Rb family of proteins also contains the
related proteins p107 and p130. We cannot eliminate a role for p107 and/or p130
inactivation in conferring antiestrogen resistance, since both SVLT and PyLT bind these
proteins, and since the PVUl and dll41 mutations eliminate binding to all members of
the pr family (21, 27). Since pr is known to be phosphorylated in response to
estrogen treatment, and since its role in regulating cell proliferation is well established, it
is likely to be the major target of estrogen action. However, it would be interesting to
determine if other pr family members also contribute to estrogen/antiestrogen
sensitivity in breast cancer cells.

SVLT possesses additional functions that might contribute to antiestrogen

resistance, including the ability to bind p300 and components of the basal transcription

70

31111
app:
tent

3W3

apparatus (21, 27). It is possible that LT binding to components of the basal transcription
apparatus plays a role in promoting antiestrogen resistance, since this function maps to N-
terminal domains of SVLT that are maintained in the T1-259 mutant (46, 47). We are not _
aware of any reports that PyLT also binds to basal transcription factors, but it may do so.
In contrast, the p300 binding activity of LT is unlikely to be involved in promoting
antiestrogen resistance, since several reports indicate that C-terrninal sequences that are
missing in the T1-259 mutant are required for p300 binding by both SVLT and PyLT (48,
49). As discussed previously, the p53 binding/inactivation function of SVLT is not
required to confer antiestrogen resistance, since it is missing in both the Tl-259 and 2809
mutants, and in PyLT.

It is interesting to compare the LT domains required to overcome cell cycle
arrests in different systems. Expression of SVLT can overcome a TGF—Bl mediated G1
arrest in keratinocytes (50) and mink lung epithelial cells (51), and this effect is
dependent upon the pr-binding domain. It is also probably independent of p53 binding,
since the HPV E7 protein, which does not bind p53, has a similar effect (51). However,
the absolute requirement for a pr-binding domain, and the lack of a role for p53
binding, in promoting proliferation is not universal. For example, at least four
independent functions of SVLT, including pr binding, p53 binding, and an unidentified
function abolished by the 8189N mutation, contribute to its ability to promote
proliferation in serum starved CVlP cells (33, 52). Mutations in any one of these
functions, including pr binding, had little effect on LT’s ability to promote proliferation
in this system. In contrast, double mutants, including pr binding/p53 binding double
mutants, showed significant defects. This variability in the requirements for pr and/or
p53 inactivation in overcoming cell cycle arrests may be due to differences in the
mechanisms by which treatments such as ICI, TGF-Bl and serum starvation induce cell
cycle arrests, or may represent differences in the requirements for pr and/or p53

inactivation between cell lines.

71

. We have also considered the possibility that smallT antigen (ST) plays a role in
conferring antiestrogen resistance, since the SVLT encoding vectors used in these
experiments contain the entire early region of the viral genome, and therefore also encode
ST. The PVUl mutant encodes a wild type ST, yet it is completely defective in
promoting proliferation in the presence of ICI. Thus, ST expression is not sufficient to
confer antiestrogen resistance. In addition, the CMVLT vector used to express PyLT in
transient transfections contains a LT cDNA, and does not encode ST. This plasmid
confers ICI resistance to MCF—7 cells with the same efficiency as SVLT, indicating that
ST expression is not required.

We attempted to determine whether LT expression confers antiestrogen resistance
to MCF—7 cells in long term proliferation assays. Although overexpression of cyclin D1
conferred antiestrogen resistance to MCF-7 cells in short term assays, it was not
sufficient to promote the long-term growth of these cells in the presence of antiestrogen
(15). Stably transfected cells expressing PyLT were resistant to ICI treatment, as
demonstrated by the facts that they contained cyclin A and hyperphosphorylated pr,
and synthesized DNA in the presence of ICI. However, this resistance was not complete.
As shown in Figure 2.5B, both pr phosphorylation and cyclin A protein levels were
reduced somewhat by ICI treatment of WT] and WT5 cells, although to a lesser extent
than in the control WT4 or d1141 cells. Similar effects were seen on BrdU and 3H-
thymidine incorporation (data not shown), suggesting that proliferation of PyLT
expressing MCF-7 cells is not completely resistant to antiestrogen treatment. However,
when the stably transfected cell lines were examined for PyLT expression by
immunofluorescence, only a subset of cells had detectable levels of LT, even during
selective growth in G418 (data not shown). The apparent partial ICI-sensitivity seen in
these experiments may therefore be due to variable expression of PyLT in the transfected
cells, rather than to an inability of LT to confer long-term antiestrogen resistance.

Finally, the fact that both pr inactivation and cyclin D1 overexpression promote

72

antiestro
might
pieces
examin
to date
a signi
failure
depen
pr s

resist

antiestrogen resistance in MCF-7 cells suggests that deregulation of the pr pathway
might occur during the development of antiestrogen resistance in viva. Several indirect
pieces of evidence support this suggestion. Although 20-25% of breast cancer cell lines
examined lack functional pr, all estrogen-responsive breast cancer cell lines identified
to date contain wild type pr (53, 54). In addition, in one study of primary breast cancers
a significant correlation was observed between the absence of pr expression and a
failure to respond to antiestrogen therapy (55). Together, these data suggest that hormone
dependence cannot be maintained in the absence of pr function, and that changes in
pr status or regulation could beiinvolved in the progression of tumors to antiestrogen

resistance.

73

10.

11.

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80

CHAPTER 3

Abrogation of Polyoma LT Induced Proliferation of Antiestrogen Treated MCF-7

Cells by p21Wan but not by pitsmm

ABSTRACT

Estrogens increase the proliferation of MCF-7 cells, a human breast cancercell
line, by activating Cdk4 and Cdk2 and this is accompanied .by pr phosphorylation. pr
is a substrate for these kinases, and its phosphorylation de-represses the transcription of
cyclin E and cyclin A, both positive regulators of Cdk2. It is not known if estrogens
regulate Cdk2 activity via pr inactivation or in a pr independent manner. Previously,
we had demonstrated that inactivation of pr using viral large tumor antigens caused
proliferation of antiestrogen-treated MCF-7 cells. Using p161NK4A and p21wa“, inhibitors
of Cdk4 and Cdk2 respectively, we demonstrate here that Cdk2 but not Cdk4 is required
for proliferation in cells where pr is inactivated by Polyoma LT. Furthermore, we
demonstrate that expression of PyLT induces Cdk2 activity in antiestrogen treated MCF-
7 cells. These results support a model in which estrogen increases Cdk2 activity by

regulating pr phosphorylation.

81

INTRODUCTION

Approximately 40% of all human breast cancers are estrogen dependent for
proliferation (1). MCF-7 cells are widely studied as a model of hormone dependent
human breast cancers. Their proliferation is stimulated by estrogens and is inhibited by
antiestrogens such as tamoxifen and ICI 182,780, in vivo as well in vitro (2-4). Estrogens
stimulate MCF-7 proliferation by regulating multiple components of the cell cycle
regulatory machinery that lead to activation of cyclin dependent kinases (Cdks), Cdk4 ‘
and Cdk2. These Cdks phosphorylate and thereby inactivate tumor suppressors of the
retinoblastoma family (5). The pr family of proteins negatively regulate the transition
from G1 to S phase by sequestering the E2F family of transcription factors (6). MCF-7
cells have wild type pr (7), and estrogen treatment of these cells arrested in G1 by
estrogen deprivation or antiestrogen treatment leads to phosphorylation and inactivation
of the pr family of tumor suppressors, E2F release and progress into S phase (8, 9).

We have previously demonstrated that inactivation of pr family members by
SV40 large tumor (LT) and Polyoma LT virus (LT) antigens causes proliferation of
antiestrogen arrested MCF-7 cells (10). This ability of LTs to induce proliferation of
antiestrogen treated cells was dependent upon an intact pr binding domain.
Furthermore, the ability of SV40 LT to overcome the cell cycle arrest resided within the
N terminal 136 amino acids of the T antigen (unpublished results), thereby excluding a
role for p53 binding or a host of other functions in this large multifunctional protein, in

overcoming an ICI arrest. Together, these results suggest that inactivation of pr family

82

members is required for and may be sufficient to overcome the antiestrogen mediated
arrest.

Both Cdk4 and Cdk2 can phosphorylate pr, and both of their activities are
regulated by estrogen and antiestrogens in MCF-7 cells (11). However, it is unclear
whether both are upstream of pr, or whether Cdk2 is activated in a pr dependent
manner (See Figure 3.1.). In order to distinguish between these two models, we tested
whether Cdk4 and Cdk2 activities were required in cells in which pr family members
were inactivated by LT. Further, we tested if the activities of these kinases were still
regulated by antiestrogens. The results indicated that Cdk4 activity is not required for
proliferation of MCF-7 cells in which pr has been inactivated by LT expression, but is
required in cells with intact pr. In contrast, Cdk2 activity is required for proliferation
irrespective of the pr status of cells. These results indicate that Cdk4 is mainly required
for in pr inactivation while Cdk2 has targets in addition to pr that are required for
inducing proliferation. Additionally, using a stable cell line that inducibly expresses LT,
we directly demonstrate that Cdk2 is activated upon LT expression in ICI treated MCF-7
cells. These results indicate that in the absence of pr function, Cdk2 activity is not fully

inhibited by antiestrogens in MCF-7 cells.

83

ICI ICI

l

Cdk4 Cdk4 Cdk2
* v e
pr pr
E2F """" 1”; E21:
l ’,Cdk2 l
l”
G" S phase 6.1- S phase
MODEL 1 MODEL 2

Figure 3.1. Models for the regulation of Cdk4 and Cdk2 activities by antiestrogens. In
Model 1, ICI regulates Cdk4 and thereby indirectly regulates Cdk2 activity in a pr
dependent manner. The predictions of this model are that Cdk4 would be redundant in cells
where pr is inactivated while Cdk2 would be still required. In addition, Cdk2 activity
would not be inhibited by antiestrogens in cells where pr is inactivated. In Model 2, both
Cdk4 and Cdk2 activities are independently regulated by antiestrogens via a pr
independent mechanism. In this case, both would be redundant and not required for
proliferation in cells in which pr is inactivated.

84

MATERIALS AND METHODS

Cell Culture. MCF-7 cells were obtained from Michael Johnson at Lombardi Cancer
Center and were routinely maintained in IMEM medium (Biofluids) containing 5% fetal
bovine serum (FBS) (HyClone), Penicillin (100 units/ml), Streptomycin (100 rig/ml). The
PyLT inducible cell lines were routinely maintained in the selective media containing

Hygromycin 10 ug/ml plus G418 (50 rig/ml).

Cell Cycle Analysis. Cells were trypsinized, washed in phosphate buffered saline (PBS),
suspended in PBS+10% FBS, fixed with 80% ice cold ethanol and stored at —20'_J C. Prior .
to analysis, cells were washed twice with PBS, then suspended in PBS + 1 mg/ml
RNaseA, 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 FACS Vantage flow cytometer; cell cycle

distribution was determined using WCycle software.

Plasmids and Cloning. Plasmids encoding the wild type LT and pr binding mutant
(Rb-LT) were obtained from Dr. Brian Schaffhausen and are described previously (12).
The pGFP CMV vectors encoding the p16INK4A and p21wan as GFP fusion proteins were
obtained from Dr. Steve Weintraub, Washington University, Saint Louis, MO. LT \cDNA
was cloned into the pLH-Zl2-I vector in a two-step ligation. The LT cDNA was excised
from the pCMV LT vector using SalI and BamHI and ligated into SalI and BamHI
digested pIC19H vector to generate the pICl9H-LT vector. In the second step, the LT

cDNA was excised from the pICl9H-LT using SalI (site was filled in with nucleotides

85

using T4 DNA polymerase to generate blunt ends) and ClaI, and ligated into the
polylinker region of pLH-Z12-I vector that was digested with EcoRI (site filled in with
nucleotides using T4 DNA polymerase to generate blunt ends) and ClaI, to generate the

pLH-Zl2-LT.

Construction of stably transfected cell lines expressing LT from an inducible
promoter. A novel gene regulation system based on small molecule regulated protein
dimerization, consisting of two plasmids and a dimerizer AP1510 was used (ARLAD
pharmaceuticals) (13). The first plasmid (pCEN-F3p65/ZlF3/Neo) contains 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 (AP1510). In
the absence of the dimerizer, 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. A cell
line, MCF-7-6, that was stably transfected with pCEN-F3p65/ZlF3/Neo was established
in our lab (C. J. Zhang unpublished results). The second plasmid (pLH-Z12-I) contains a
hygromycin resistance gene, and a polylinker downstream of a promoter that contains
DNA elements that are recognized by the DNA binding domain encoded by pCEN-
F3p65/ZlF3/Neo. The pLH-Zl2-I—LT vector was stably transfected into the MCF-7-6
cell line using the Lipofectin reagent (Gibco BRL) and hygromycin resistant clones were

selected (30 rig/ml) and then screened by immunoblotting and immunofluorescence.

86

Chemicals and Antibodies. ICI 182,780 was a gift of A. Wakeling (Zeneca
Pharmaceuticals) and 17B-estradiol (E) was purchased from Sigma. 5’
Bromodeoxyuridine was obtained from Boehringer Mannheim. AP1510 was kindly
provided by ARIAD Pharmaceuticals (Cambridge, MA). LT antibody was a rat
polyclonal and was a kind gift of Dr. Michelle Fluck. Cdk4 antibody was a goat H-22
antibody from Santa Cruz Biotechnology, whereas the Cdk2 antibody was a rabbit
polyclonal gifted by Charles J. Sherr. Antibodies to cyclins A, D1, E, the kinase

inhibitors p21wan and p27Kipl and actin are described in the following chapter.

Immunofluorescence. MCF-7-6 cells were plated on glass coverslips and transiently
transfected with either the LT plasmid alone or in combination with the variouspr
plasmids using the Superfect transfection reagent (Qiagen). After transfection, cells were
treated with medium containing 5% charcoal stripped serum and ICI (100 nM) and were
labeled with 25 11M 5—Bromo-2’-deoxyuridine (BrdU) for 5 hours before fixation at 48
hours post transfection. The double immunofluorescence procedure for detecting
transfected proteins and BrdU has been described previously (10). BrdU incorporation
was detected using the appropriate or-BrdU antibody (mouse or-BrdU). Cells were viewed
under a fluorescent microscope (Olympus), and the percentage of LT expressing cells
that were positive for BrdU, and of cells that were positive for BrdU alone was
determined. At least one hundred LT positive and negative cells were counted in each
experiment. For the detection of LT expression in the stable inducible cell lines, cells
were plated on glass coverslips and were treated with AP1510 or untreated and harvested

24 hr after treatment. Imunofluorescence for LT was done as described above and cells

87

were viewed under a fluorescent microscope. The images shown in Figure 3.3B. were
taken on the Olympus fluorescent microscope, scanned and converted to grayscale using

the Adobe Photoshop software program.

Long Term Growth Assays. Cells were plated at 20,000/well on a 24 well tissue culture
plate and after overnight incubation in FBS containing media, they were kept in CSS
containing media with 100nM ICI for 48 hr to induce a G0/Gl arrest. The media was
then changed to either CSS, CSS plus ICI or CSS plus E alone or to these media plus
AP1510 (300nM). Cells were harvested every 2 or 3 days and fresh media with the
appropriate treatments added every 2 or 3 days for the duration of the experiment. All
samples were harvested in triplicate and the DNA content determined by a fluorometric

assay described previously ( 14).

Immunoprecipitations and Kinase Assays. Immunoprecipitations (IP) and Cdk4
activity measurements were performed using a modification of a published method (15).
Unless indicated otherwise, all manipulations were carried out on ice. Cells were washed
twice in ice cold PBS, harvested by scraping in PBS and pelleted; the pellets were frozen
and stored in liquid nitrogen until the day of analysis. Cells were resuspended and lysed
by sonication in IP buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 2.5
mM EGTA, 0.1% Tween-20, 10% glycerol, 1 mM DTT, 10 mM B-glycerophosphate, 1
mM NaF, 1mM NaVO4, 0.1 mM PMSF, 10 pg/ml leupeptin, and 2 ug/ml aprotinin). The
lysates were cleared by centrifugation, and protein concentrations were quantitated using

BioRad’s protein assay reagent. For IPs, 75 ug of total protein was diluted to 500 pl with

88

[P buffer, then incubated for 60 minutes (m) rocking at 4° C with 1.5 pg antibody (anti-
Cdk4 H-22-G or normal goat IgG, Santa Cruz or anti-Cdk2 antiserum) bound to 7.5 pl
protein G agarose beads (Boehringer Mannheim). Beads were pelleted and washed four
times with 100 pl IP buffer and twice with 100 pl kinase buffer (50 mM HEPES pH7.5,
10 mM MgC12, 1 mM DTT, 2.5 mM EGTA, 10 mM B-glycerophosphate, 0.1 mM
NaVO4, and 1 mM NaF). Pellets were then suspended in 40 pl kinase buffer containing
10 pCi y-(32P) ATP, 20 pM cold ATP, and 1.0 pl glutathione sepharose 4B (Amersham
Pharmacia) to which approximately 2.0 pg of a fusion protein between glutathione-S-
transferase (GST) and amino acids 792 to 928 of human pr were bound (GST-Rb
bacterial expression vector was kindly provided by Dr. William Kaelin of the Dana
Farber Cancer Institute) or Histone H1 (HHI) was used for Cdk2 assays. The reactions
were incubated at 30° C for 30 m with occasional mixing, after which they were boiled in
SDS loading buffer containing mercaptoethanol. Beads were pelleted and the
supematants transferred to clean tubes. Aliquots of the reaction products were resolved
on 10% SDS polyacrylarnide gels, which were then dried and exposed to autoradiography
film. Phosphorylated GST-Rb or HHI bands were quantitated by phosphorimaging with

a Storm phosphorimager (Molecular Dynamics) using ImageQuant software.

89

RESULTS

LT induced proliferation is sensitive to p21wm but resistant to pl6mx4" Cdk
inhibitor. A requirement for the pr binding domain of PyLT antigen for conferring
antiestrogen resistance was confirmed in initial experiments. Plasmids encoding 'wild
type or a pr binding mutant (Rb- LT) of PyLT were transiently transfected into MCF-7
cells and the cells were treated with antiestrogen ICI 182,780 for 43 hr and then BrdU (25
pM) labeled for an additional 5hr before fixation. The fixed cells were analyzed for PyLT
expression and BrdU incorporation by indirect immunofluorescence. The percentage of
cells that were actively synthesizing DNA was determined in PyLT expressing cells and
in untransfected cells (Figure 3.2A upper panel). The results of this experiment
demonstrated that expression of PyLT caused antiestrogen resistance. The pr binding
domain of wild type PyLT was absolutely required for this effect, as shown by the
inability of the Rb-LT mutant to increase proliferation above the levels observed in
untransfected cells despite similar expression levels of both proteins (Figure 3.2A, lower
panel). These results confirmed previous results obtained with SV40 LT (10).

To determine if cells in which pr family members were inactivated by LT
required Cdk4 and Cdk2 activities for proliferation, MCF-7 cells were transfected with
PyLT alone, or together with a vector expressing either Green Fluorescent protein (GFP),
p16INK4A-GFP or p21wan-GFP. p16INK4A is a specific inhibitor of Cdk4 (6) whereas

W
18“

p2 inhibits both Cdk2 and Cdk4, although recent reports suggest that it may also

facilitate active cyclin D-Cdk4 complex formation (16). These inhibitors were expressed

90

Figure 3.2. MCF-7 cells expressing PyLT are sensitive to p21wan but refractory to
p161NK4A arrest. A) MCF-7 cells were transfected with wild type PyLT or a Rb binding
mutant (Rb-) of PyLT, treated with ICI (IOOnM), and proliferation was assayed by
double indirect immunofluorescence for PyLT and BrdU at 48 hr post transfection as
described in Materials and Methods. The percentage of in PyLT positive and negative
cells that were aslo BrdU postive cells is shown as mean +/- SE. of at least three
independent experiments. At least 100 LT positive and negative cells were counted in
each experiment. In a parallel experiment, wild type and mutant PyLT expression Was
assessed by Western blotting (lower panel, UT-untransfected control). B) MCF-7 Cells
were cotransfected with PyLT plus either pGFP (vector), pl6-GFP or p21-GFP encoding
plasmids. Proliferation in the presence of ICI was assayed by double indirect
immunofluorescence for LT and BrdU( I LT Positive cells, D LT negative cells ) C)
The Rb binding mutant (Rb-LT) of PyLT was cotransfected with the above GFP
constructs in cycling MCF-7 cells, and proliferation was assayed as described above.

91

% BrdU+ cells

% BrdU+ cells

% BrdU+ cells

30-1

20‘

10"

 

 

Rb- LT PyLT
UT PyLT Rb-LT

u t ’ " ‘1 "L -‘J ." '3' ‘
. .. .fn’of - “n.7- "L a "5 ‘LI 1' r. ft:
m an... Py I , I

 

 

 

21.1.. _.

PyLT PyLT PyLT PyLT
+GFP + p16 + p21

 

 

 

 

 

 

Rb-LT Rb-LT Rb-LT Rb-LT
+GFP + p16 +p21

92

 

 

CSS+ICI

 

 

 

IT+
DT’

 

 

 

 

 

 

 

 

 

 

 

CSS +ICI
IT+
C] T-
CSS +E
IT+
DT-

as fusion proteins with GFP to facilitate their detection by visual inspection under a
fluorescent microsc0pe. In these cotransfection experiments, a higher percentage of cells
expressed the GFP fusion proteins (IS-20%) compared to PyLT (~5%). Therefore, it was
likely that cells expressing PyLT would also express the GFP fusion proteins. The ability
of these proteins to prevent PyLT mediated proliferation of ICI treated cells was assessed
by double indirect immunofluorescence for PyLT and BrdU as described above for
transfections with PyLT alone. In control experiments, Rb-LT was cotransfected with
pGFP, p16mK4A-GFP or p21wan-GFP in 17B-estradiol (E) treated MCF-7 cells to
determine if p16INK4A or p21wafl inhibited estrogen induced proliferation of MCF-7 cells
where Rb was functional. The results shown in Figure 3.2B and 3.2C demonstrate that
cells expressing the pGFP vector exhibited a twofold decrease in proliferation relative to
untransfected cells treated with estrogen or to those transfected with PyLT alone. Thus,
GFP alone inhibits proliferation, but not down to levels observed in cells treated with ICI.
The level of proliferation in GFP cotransfected cells therefore served as a baseline to
assess the effects of p16INK4A and p2lw‘1fl on the inhibition PyLT induced proliferation.
The Cdk2 inhibitor, p21walfl inhibited proliferation in PyLT expressing cells while the
Cdk4 inhibitor, pl6INK4A did not (Figure 3.2B). However, in control transfections with
Rb-LT, p16'NK4A inhibited proliferation as efficiently as p21wafl (Figure 3.2C). These
results demonstrate that Cdk4 activity is not required in cells in which pr has been
inactivated, but is required in cells with intact pr. However, Cdk2 activity is required
regardless of the pr status of cells. These results also indicate that Cdk4 is required for
pr inactivation, while the Cdk2 has targets in addition to pr. Finally, they suggest that

antiestrogens do not. inhibit Cdk2 activity if pr family members have been inactivated

93

A. LT ( +AP)

 

   

LT-6 cell line
— AP1510 + AP1510

Phase
Contrast

 

Figure 3.3. Selection of stable cell lines that express PyLT in an inducible manner. A)
The MCF-7 derivative (7-6) was transfected with the plasmid expressing PyLT from an
inducible promoter (pLH-ZlZ-I-LT), and clones were selected in Hygromycin (30
pg/ml), and screened for PyLT expression by Western blotting after 24 hrs of AP1510
(300nM) (+AP) treatment (top panel). Three clones exhibiting high expression were then
tested for expression in the absence and presence of AP1510 (bottom panel). B) One
clonal cell line, LT-6, was analyzed for PyLT expression by indirect
immunofluorescence. The figure shows immunofluorescence for PyLT in the absence (-
AP1510) and presence (+AP1510) of AP1510, along with a phase contrast picture of the
same fields. The Fluorescent images were converted to grayscale using the Adobe
Photoshop software program.

94

Screening PyLT inducible cell lines. To test whether Cdk2 activity is induced by PyLT
expression, and to address the mechanism of its induction, we constructed stable
inducible cell lines in which the expression of PyLT was regulated by the addition of a
small molecule dimerizer, AP1510. The PyLT encoding cDNA was cloned into a vector
downstream of an AP1510 inducible promoter. The description of the inducible plasmid
system and the selection of stable cell lines is described in Materials and Methods.
Briefly, MCF-7-6 cells were transfected with a vector expressing PyLT from- the
inducible promoter, and hygromycin resistant clones were selected and expanded into cell
lines.

These clones were screened by Western blotting (Figure 3.3A), and several with low
basal and high induced levels of PyLT induction upon the addition of AP1510 were
chosen for further characterization. LT-5, LT-6 and LT-21 were three clonal cell lines
used for further studies (Figure 3.3A lower panel and data not shown). The expression of
PyLT in the inducible cell lines was also examined by immunofluorescence. Significant
variability in the expression of PyLT was observed upon AP1510 treatment (Figure
3.3B), ranging from undetectable to very high levels. This variability in expression was
observed for all three clonal cell lines tested. Approximately 50-60 % of the cells

expressed detectable PyLT even in the continued presence of the selective agent,

hygromycin (10 pg/ml).

PyLT induces proliferation of ICI treated cells. Initially, the effects of PyLT
induction on cell cycle progression of cells arrested in G0/Gl by ICI treatment were

assessed in LT—6, LT-S and LT-21 cell lines. The results from one experiment in LT-S

95

 

 

 

 

 

 

40 - +E
2 30 ‘
g +ICI+
a 20 4 ‘P
E
m 10 _. +|Cl
e“

0 j I l

0 12 24 36

Time after Treatment (hrs)

m E MM?
'0 612 24361512 24 381512 24 315m

 

----mn

‘ ‘ #1:: ”'1" .-"‘-'TTé-ECY°“" A

1.

- cud-u--- - u QQ~ujCyclin E

~-._--..~~.V---.-~-— CYClinDl
~-h“”-~ "“””pfl

g". ‘. .. .u" ..‘.'-'.. .."..'-.‘.-.~~ L- - _- .. - ..', .
>~~~.--*.-—“-—-~~"ACIIH

Figure 3.4. PyLT induces S phase entry in ICI treated MCF-7 cells in short term assays.
A) LT-5 cells were growth arrested in ICI for 48 hours, treated with CSS+ICI, CSS+E or
CSS+ICI+AP, then harvested at 12 hr intervals and the cell cycle profile was analyzed by
flow cytometry. The percentage of cells in S phase at the various time points after the
indicated treatments is shown. Results represent the average +/- SE. of an experiment
done in triplicate. B) In a parallel experiment, LT-S cells were treated as in (A) above.
They were harvested at the time points indicated, and cell extracts were analyzed by
western blotting(_for PyLT, cyclin A, cyclin B and cyclin D1, and the Cdk inhibitors
p21wa“ and p27 '1’]. Actin was probed as a loading control

96

cells is shown in Figure 3.4A. LT-5 cells were pre-arrested with ICI (10nM) for 48 hrs,
and were then treated in one of the following ways. PyLT expression was induced by the
addition of AP1510 (300nM) in the continued presence of ICI, or cells were treated with
ICI alone or with E (10 nM) to serve as negative and positive controls respectively. Cells
were harvested at 12 hr intervals after treatment, and analyzed by flow cytometry to
determine their cell cycle phase distribution. Expression of PyLT caused an increase in
the percentage of cells in S phase in ICI treated cells between 12 and 24 hours, from 6—7
% in S phase at the 0 hr time point to 20 % cells in S phase by 24 hr (Figure 3.4A).
Similar results were obtained for the LT-6 and LT-21 cell lines (data not shown). In
comparison, cells treated with E showed a much higher percentage of cells in S phase
(upto 40 %) at the 24 hr time point. These results demonstrate that induction of PyLT
expression increased proliferation of ICI treated cells though not to as high a level as E
treatment and qualitatively confirms the results obtained in the transient transfection
experiments.

To investigate the effects of PyLT expression on cell cycle components that might
contribute to antiestrogen resistance, cells were harvested at the given time points after
AP1510 treatment in an experiment performed in parallel to the one shown in Figure
3.4A. They were analyzed by Western blotting for the expression of PyLT and cell cycle
regulatory proteins including cyclins A, E and D1 and the kinase inhibitory proteins,
p21walfl and p27Kip' (Figure 3.4B). Actin was used as a loading control. A clear induction
of PyLT protein was observed by 6 hours after AP1510 addition, and high levels were
maintained for the duration of the experiment. Cyclin A protein levels increase at the Gl-

S phase transition making this protein a marker for S phase progression. An increase in

97

cyclin A protein levels was observed by 24 hrs after AP1510 treatment. However, the
levels of cyclin A induced were lower than in cells treated with E at the corresponding
time points, which correlated with the cell cycle data. Cyclin D1 and cyclin E did not
show any obvious increase at the 6 or 12 hrs time point upon LT induction. However, at
the later time points, E treated cells showed a decline in cyclin E. Since cyclin E is a G1
cyclin that is degraded during late S phase (17), this decrease is likely a consequence of
cell cycle progression. A similar decline in cyclin D1 levels was noted in all treatments at
the 24 and 36 hr time points. One possible explanation for this result in E treated cells is
that the levels of cyclin D1 are downregulated in S phase (6). Treatment with ICI has also
been reported to decrease the levels of cyclin D1 and could account for the decreases
seen in cells treated with ICI alone (18). The reasons for the decline in cyclin D1 levels in
cells treated with ICI plus AP are less clear, and either mechanism could be responsible.
In any case, the levels of cyclin D1 or cyclin E were not elevated by AP treatment, and
thus could not explain the increase in the percentage of cells in S phase in PyLT

expressing cells. The levels of the kinase inhibitor, p21wan

, were lower in PyLT
expressing cells compared to cells treated with ICI alone, though but not as low as seen in
E treated cells, suggesting that changes in the levels of p21wan might be contributing to

the S phase progression upon PyLT induction. The levels of p27mp' did not change

significantly upon PyLT induction.

PyLT induces both Cdk2 and Cdk4 activities in ICI treated MCF-7 cells. In order to

determine the mechanism(s) by which PyLT expression induced proliferation of ICI

98

.3’

50

.01
30..
20*
10]

o ‘17 l I

O 24 48 72 96 120

% 8 Phase

 

 

 

Time (hours)
B.
__+Ig:ar+ +10
0 24 48 72 96 24‘ hr
~ ~ ~ I. PyLT
m ,..;.1 M Cyclin A

CyclinDl

 

.11.,“ - .. .;..-_._J 1,1.“ mm . .. 1‘ p21

”WWMWM Adi“

Figure 3.5. PyLT induces proliferation of ICI treated cells over 2 cell cycles. A) LT-6
cells were growth arrested in ICI for 48 hours, then treated with CSS, CSS+ICI,
CSS+AP, CSS+E, CSS+AP+E or CSS+ICI+AP. Cells were then harvested at 24 hour
intervals and the cell cycle profile was analyzed by flow cytometry. The percentages of
cells in S phase at various time points after the indicated treatments are shown. B) LT-6
cells arrested as described in (A), were treated with AP1510; then analyzed for PyLT,
cyclin A, cyclin D1 and p21wm and p27Klpl levels by immunoblotting. A time matched
control (24 hr*) of ICI treated cells, was included. Actin blots were done to confirm equal
loading.

99

A, +lCI+AP ICI E E
l l I gG
0 24 48 72 96 24 24 24 hr

 

Cdk4
activity
“ 4— GST-Rb

CDK4 Actlvlty In LTB Cells

 

 

on 24h 48h 72h 96h 24h 24h 24h
ICI E E,lgG

+ICI+AP ICI E E
B . l l I gG
0 24 48 72 96 24 24 24 hr Cdk2

' m -' ” 3* -‘ "1 activity

CDK2 Activity In LTO Coll.

 

    

 

 

0h 24h 48h 72h 96h 24h 24h 24h
ICI E E.
lgG

Figure 3.6. PyLT induces Cdk2 and Cdk4 activities in ICI treated MCF-7 cells. LT-6
cells were growth arrested in CSS+ICI (10 nM) containing media for 48 hrs. After 48 hrs,
PyLT was induced by AP1510 (300 nM) treatment of cells in ICI containing media. Time
matched controls (24 hr) where only ICI was added, and one with E (10 nM), were
included as negative and positive controls, respectively. Cell extracts were prepared at 24
hr intervals and were analyzed for A) Cdk4 (GST-Rb as substrate) and B) Cdk2 (Histone
H1 as substrate) in vitro kinase activity as described in Materials and Methods. Normal
pre-immune serum was used as negative control for the kinase assays (IgG). The kinase
activity was quantitated by phosphorimager scanning and is shown graphically below the
respective autoradiographs. These results are representative of two independent
experiments.

100

treated cells, LT-6 cells were prearrested with ICI (10 nM) and then either treated with
ICI, E or CSS individually or in combination with AP1510. Cells were harvested at 24 hr
intervals for 4 to 5 days and were analyzed for cell cycle distribution. Results are shown
in Figure 3.5A. Py LT expression caused an increase in the percentage of cells in S phase
to a similar extent both in the absence of E and the presence of ICI. However, the
percentage of S phase observed in the E or E plus AP treated cells was approximately two
fold higher than in PyLT expressing cells and is similar to the results described in the
previous section for the LT-S cell line. The fact that E and E+AP treated cells showed
similar levels of proliferation indicated that PyLT expression did not interfere with E
stimulated proliferation and was not toxic to these cells.

In a parallel experiment, ICI arrested cells were induced with AP1510, harvested
at every 24 hr intervals for 4 days and were subjected to Western blotting and in vitro
kinase assays for Cdk4 and Cdk2 activity. ICI and E treated cells were harvested 24 hrs
after treatment and served as negative and positive controls, respectively. As noted
previously, the increase in cyclin A protein at the 24 and 96 hr time points corresponded
with the increase in the percentage of cells in S phase. Cdk2 activity was clearly induced
at 24 hr after AP1510 treatment of ICI treated (Figure 3.6B). However, E treated cells
showed approximately two-fold higher Cdk2 activity at the 24 hr time point, which
correlated with the increased percentage of cells in S phase in the E treated cells. Thus,
PyLT expression causes activation of total Cdk2 activity that closely parallels the
induction of cyclin A protein and S phase entry in ICI treated MCF-7 cells. Surprisingly,
Cdk4 activity was also induced at the 24 hr time point after PyLT induction (Figure

3.6A).

101

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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2’ + E
‘g + ICI+AP
‘g’ 1000 - +AP
O
E - CSS
0 + ICI

100
0 3 6 9 12
DAYS

Figure 3.7. PyLT confers partial antiestrogen resistance in long-term assays. PyLT
inducible cells (LT-6) were growth arrested in CSS+ICI containing media for 48 hrs. The
media was then changed to CSS+ICI, CSS alone or CSS+E or to these media plus
AP1510 (300nM). A) Cells were harvested every 2 or B) 3 days. Fresh media with the
appropriate treatments were added every 3 days for the duration of the experiment. All
samples were harvested in triplicate and the DNA content per well in nanograms (ng) was
determined by a fluorometric assay and is represented as mean +/- SE. The results from
two independent experiments are shown. Note that graph B. is plotted on a logarithmic
scale to show the small increase in proliferation upon PyLT expression. This increase is
difficult to see on a linear scale due to the large differences in proliferation between E
and ICI treated cells in this experiment.

102

These results demonstrate that ICI cannot completely inhibit Cdk4 or Cdk2 activities in

MCF-7 cells in which pr function is abrogated by LT.

Effect of LT induction on Long Term Growth of ICI treated MCF-7 cells. To assess
the effects of pr inactivation on the long-term growth of MCF-7 cells, proliferation
assays were carried out. LT—6 cells were plated on 24 well plates, prearrested with [Cl
and treated as described in Materials and Methods. Cells were harvested every 2-3 days
over a period of 8 to 12 days and the DNA content measured by a fluorometric assay. The
results from two independent experiments are shown graphically in Figure 3.7. They
demonstrate that expression of PyLT enhanced the proliferation of cells treated with ICI
above the levels seen in cells treated with ICI or depleted of E in the long term and are
qualitatively similar to the short-term results. Thus inactivation of pr leads to at least
partial antiestrogen resistance in the long term However, in the long-term, the
proliferation upon PyLT expression was much lower than that seen in the short-term
assays suggesting that events in addition to pr inactivation may be required to confer
complete antiestrogen resistance in the long term. The possible reasons for this are

discussed below.
DISCUSSION
The Rb gene is mutated in a significant percentage of breast cancers (19, 20) and

a much larger percentage of breast cancer patients show alterations in the pr pathway

resulting from overexpression of cyclin D1 or loss of p16INK4. Significantly, estrogens

103

regulate proliferation of estrogen responsive human breast cancer cells by activating
Cdk4 and Cdk2, which phosphorylate and thereby inactivate pr (8, 9). However, the
contribution of pr inactivation to the development of antiestrogen resistance, a
significant problem in breast cancer therapy, is not understood. We previously
demonstrated that expression of SV40 and polyoma viral LT antigens caused antiestrogen
resistance in MCF-7 cells, and that the pr interaction domain of these T antigens was
required for conferring antiestrogen resistance (10).

Since both Cdk4 and Cdk2 can contribute to pr inactivation, and their activities
are inhibited by ICI, it was unclear if antiestrogens regulate Cdk2 activity via a pr
dependent or independent mechanism (See Figure 3.1). This is important to determine,
because if ICI regulates the activities of both Cdk4 and Cdk2 independently, deregulation
of both would be required for tumors to acquire antiestrogen resistance. We initially
determined if both Cdk4 and Cdk2 were required for proliferation in cells where pr was
inactivated and Cdk2 activity was regulated by ICI under these conditions. Using
inhibitors of Cdk4 and Cdk2, we demonstrate that Cdk2 but not Cdk4 kinase activity is
required for proliferation in cells where pr family members are inactivated. In addition,
using stable inducible cell lines, we showed that both Cdk4 and Cdk2 activities are
induced by LT expression in the presence of the antiestrogen, ICI. Further, we confirm
that PyLT expressing cell lines proliferate in the presence of ICI, at least in the short
term.

Reports have suggested that estrogen activates cyclin E-Cdk2 complexes by
inducing cyclin D1 protein expression, resulting in the redistribution of p21waf', from

these complexes to cyclin D1-Cdk4 complexes, and additionally by decreasing the levels

104

 

of p21wa“ (8, 9, 21). The results presented here indicate that total Cdk2 kinase activity is
induced upon PyLT expression in ICI treated cells without any increase in cyclin D1
levels. Although we have not specifically tested whether cyclin E-Cdk2 activity is
induced upon PyLT induction, our results demonstrate that total Cdk2 activity is partially
regulated by ICI in a pr dependent manner. The observed increase in Cdk2 activity in
ICI treated cells upon PyLT expression could be explained by the increase in cyclin A
protein levels (Figure 3.5B). Since the cyclin A promoter has been reported to be
regulated in a E2F dependent manner, and cyclin A-Cdk2 activity is essential for G1 to S
phase transition (22, 23), release of E2F upon pr inactivation could explain the
upregulation of cyclin A, the increase in Cdk2 activity and proliferation. Alternatively,
cyclin A-Cdk2 complexes could titrate p21wm and lead to cyclin E-Cdk2 activation.

The mechanism of activation and the downstream targets of Cdk4 and Cdk2 have
been under study for some time. pr has been identified as the major physiological target
of Cdk4 (15). Cdk4 activity is redundant in cells where pr has been inactivated,
suggesting that the only role for Cdk4 is the inactivation of pr (24). Thisis consistent
with our result that the specific Cdk4 inhibitor, pl6INK4A, did not prevent the proliferation
of cells transfected with PyLT, while it did prevent proliferation of cells transfected with
the pr binding mutant of PyLT. Although Cdk4 activity was not required for
proliferation, surprisingly, it was induced by PyLT. To our knowledge, this is the first
observed regulation of Cdk4 activity by PyLT, and the mechanism of this activation
needs to be further investigated.

An interesting question raised by our studies arises from the observation that

expression of PyLT increased proliferation above the levels seen in ICI treated cells, but

105

to levels approximately two fold lower than those observed in E treated cells in the short
term assays (Figure 3.4A and 3.5A). Similar results were observed for three independent
PyLT inducible cell lines LT-5, LT-6 and LT-2l. Furthermore, this correlated with 2 fold
lower levels of Cdk2 and Cdk4 activities in PyLT expressing compared to E treated cells.
. In long-term proliferation assays, PyLT induction increased proliferation of ICI treated
cells, but to much lower levels than that observed in E treated cells. One interpretation of
these results is that pr inactivation is not as efficient as estrogen in causing
proliferation, suggesting that there are other targets of estrogen that mediate its
proliferative effects. Estrogen induces multiple components of the cell cycle machinery in
MCF-7 cells, including cyclin D1, c-Myc and Cdc25A and decreases the levels of p21wan
and p27K1p' kinase inhibitors (5). Of these effects, c-Myc is known to promote
proliferation via a pr independent mechanism that involves activation of cyclin E-Cdk2
activity (25). Since we have measured total Cdk2 activity, it is possible that cyclin E
associated kinase activity remained low in PyLT expressing cells resulting in decreased
total Cdk2 activity and proliferation compared to E treated cells. However, cyclin E-Cdk2
activity has been shown to be required for Gl-S phase transition regardless of the pr
status of cells (26). Therefore, it would be interesting to determine if this is true in PyLT
expressing cells.

Another possible explanation for decreased proliferation upon PyLT expression is
that only cells that express PyLT above a certain threshold level are able to overcome an
ICI mediated arrest. This is consistent with the observation that transfected cell lines with
lower levels of PyLT expression, exhibited no detectable increase in the proliferation of

ICI treated cells upon induction of PyLT (data not shown). Since there appears to be

106

variable expression of PyLT as assessed by immunofluorescence (Figure 3.3B and data
not shown) with approximately 50-60 percent cells showing detectable PyLT expression,
it is possible that only cells expressing “high” levels of PyLT in the population will
proliferate in the presence of ICI.

In conclusion, the results presented in this chapter confirm that inactivation of
pr and related family members in MCF-7 cells causes partial antiestrogen resistance, at
least in the short term. This is accompanied by an induction of cyclin A protein, and total
Cdk2 activity. They confirm that the role of Cdk4 in proliferation is largely to inactivate
pr or a related family member, whereas Cdk2 has other functions that are necessary for
proliferation. Interestingly, at least a part of the regulation of Cdk2 activity by estrogen
appears to be via pr inactivation. These results support a model for estrogen mediated
proliferation in which estrogen activates Cdk4, and this leads to Cdk2 activation

indirectly by inhibiting pr function (For model see Figure 3.8).

107

ICI

l

Cdk4

v
pr

j Cyclin A
E2F /2

l {Cdk2

Ar"
G1- S phase

Figure 3.8. Model for the regulation of Cdk4 and Cdk2 activities by antiestrogen.
According to this model, ICI indirectly regulates Cdk2 activity in a pr dpendent
manner. ICI regulates the activity of Cdk4 that causes pr phosphorylation and
inactivation. Inactivation of pr releases the E2F transcription factor that increases the
transcription of cyclin A protein levels and thereby results in Cdk2 activation and
contributes to Gl-S phase transition.

108

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Foster, J. S., Henley, D. C., Bukovsky, A., Seth, P., and Wimalasena, J.
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Estrogen-induced activation of Cdk4 and Cdk2 during Gl-S phase progression is
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Varma, H. and Conrad, S. E. Reversal of an antiestrogen-mediated cell cycle
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Foster, J. S., Henley, D. C., Ahamed, S., and Wimalasena, J. Estrogens and cell-
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Amara, J. F., Clackson, T., Rivera, V. M., Guo, T., Keenan, T., Natesan, S.,
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Cheng, M., Olivier, P., Diehl, J. A., Fero, M., Roussel, M. F., Roberts, J. M., and
Sherr, C. J. The p21(Cip1) and p27(Kip1) CDK 'inhibitors' are essential activators
of cyclin D-dependent kinases in murine fibroblasts, Embo J. 18: 1571-83., 1999.
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Watts, C. K., Sweeney, K. J ., WarLTers, A., Musgrove, E. A., and Sutherland, R.
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CHAPTER 4

Antiestrogen ICI 182,780 Decreases Proliferation of IGF-1 Treated MCF-7 Cells

Without Inhibiting the PI3K/AKT Pathway.

ABSTRACT

Both estrogens and growth factors such as Insulin like growth factor-1 (IGF-l) are
potent mitogens for human breast cancer cells. Antiestrogens, including ICI 182,780
(ICI) and tamoxifen, inhibit both estrogen and IGF-1 induced proliferation of estrogen
responsive breast cancer cells. The mechanism(s) by which ICI inhibits IGF-l mediated
proliferation of MCF-7 cells, an estrogen receptor (ER) positive, estrogen responsive
human breast cancer cell line, were addressed in this study. IGF-l induces proliferation in 1
MCF-7 cells by activating phosphoinositol 3(OH) kinase (PI3K) and its downstream
target AKT, which regulates components of the cell cycle machinery including cyclin D1
protein. Numerous studies have suggested that antiestrogens regulate MCF-7 cell
proliferation by affecting the components of the IGF-l signaling pathway including IGF-
1 Receptor protein levels, Insulin receptor substrate—1 (IRS-l) protein levels and
PI3K/AKT activity. The results presented here demonstrate that ICI does not abrogate the
ability of IGF-1 to induce proliferation or cyclin D1 expression. In addition IGF-l
stimulated phosphorylation of AKT is unaffected by ICI treatment, and transient

expression of constitutively active PI3K or AKT was unable to induce proliferation in ICI

113

treated MCF-7 cells. Together, these results demonstrate that ICI does not block IGF-l
signaling, and suggest a model wherein ICI inhibits IGF-l mediated proliferation by

mechanisms other than downregulating the PI3K/AKT signaling pathway.

INTRODUCTION

Both growth factors and the sex steroid, estrogen, regulate proliferation of human
breast cancer cells. Growth factors bind to membrane bound tyrosine kinase receptors
that activate signaling cascades affecting cell cycle progression. Insulin like growth
factor-1 (IGF-l) stimulates proliferation of the MCF-7 human breast cancer cell line by
activating the membrane bound tyrosine kinase receptor, IGFl-R (1, 2). Activated IGFl-
R recruits and phosphorylates the insulin receptor substrate-l (IRS-1), which in turn
activates phosphoinositol 3(OH) kinase (PI3K), a major mediator of the IGF-l signaling
pathway (3). PIBK activity is essential for the proliferation of MCF-7 cells, as chemical
inhibitors of PI3K prevent MCF-7 proliferation (4). P13K activates downstream kinases
including AKT, p7OS6 kinase and glycogen synthetase 3 kinase (5). One of the key
downstream targets of these kinases is cyclin D1, an important regulator of cell cycle
progression (6).

Estrogens activate the estrogen receptors alpha (EROt) and beta (ERB), ligand
activated nuclear transcription factors (7) that regulate transcription of target genes. ERor
plays a critical role in breast tissue proliferation, as EROt knockout mice do not develop
breast tissue at puberty. ERfl does not appear to be important for breast epithelial

proliferation, since ERB knockout mice show normal breast development (8). MCF-7

114

cells are ERor positive (9), and their proliferation is regulated by estrogens and
antiestrogens (10-12). Evidence obtained in the past several years has led to the
development of a model to explain estradiol (E) induced proliferation of MCF-7 cells. In
this model, E induces cyclin D1 protein expression, which then activates the cyclin
dependent kinases Cdk4 and cyclin E-Cdk2. Cdk4 is activated by direct binding of cyclin
D1, whereas activation of Cdk2 involves redistribution of the kinase inhibitor, p21wa",
from cyclin E-Cdk2 to cyclin Dl-Cdk4 complexes, thereby relieving inhibition of cyclin
E-Cdk2 kinase (13, 14). Additionally, downregulation of 1321““ and p27ij1 and
activation of Cdc25A phosphatase by E, have been proposed to contribute to E stimulated
activation of Cdk2 complexes (15-17). Active Cdk4 and Cdk2 phosphorylate and
inactivate pr, a negative regulator of cell cycle progression, releasing the E2F family of
transcription factors that turn on genes important for cell cycle progression (18).
Antiestrogens inhibit proliferation of MCF-7 cells, and reverse the above mentioned cell
cycle changes induced by estrogen treatment (13, 14).

In addition to reversing the proliferative effects of estrogens, antiestrogens such as
tamoxifen and ICI 182,780 (ICI) inhibit growth factor induced proliferation of MCF-7
cells (19-21), but the mechanism(s) by which they do so are unclear. Defining them may
contribute to the understanding of antiestrogen resistance, because deregulation of growth
factor signaling pathways has been implicated in the development of antiestrogen
resistance in breast cancer patients (22, 23). Considerable evidence indicates that
antiestrogens and estrogens regulate intermediates in the IGF-l signaling pathway,
including IGFl-R (24, 25) and IRS-1 protein levels (26), and IRS-l phosphorylation (27),

suggesting that antiestrogens might inhibit proliferation by inhibiting IGF-l signaling.

115

This study was undertaken to define the molecular mechanism(s) by which ICI
inhibits IGF-l induced proliferation in MCF-7 cells. To approach this, we investigated
the effects of IGF-I on cell proliferation and the expression of cell cycle regulatory
molecules in the presence and absence of ICI. Our results demonstrate that while ICI
decreases the absolute levels of proliferation and the cell cycle regulatory molecules
including cyclin D], it does not eliminate the effects of the effects of IGF-1 on these
targets. In addition, IGF-l induced phosphorylation of AKT, a key regulator of cyclin D1
protein levels (28) was not inhibited by ICI. Finally, the expression of constitutively
active PI3K or AKT was insufficient to overcome an antiestrogen mediated growth arrest
of MCF-7 cells. Together, these results suggest a model in which ICI decreases
proliferation of IGF-I treated cells by affecting pathways other than the PI3K/AKT

pathway.

MATERIALS AND METHODS

Cell Culture. MCF-7 cells were obtained from the Dr. Michael Johnson at the Lombardi
Cancer Center and were routinely maintained in IMEM medium (Biofluids) containing
5% fetal bovine serum (FBS) (HyClone), penicillin (100 units/ml) and streptomycin (100
pg/ml). For serum free experiments, MCF-7 cells were trypsinized, resuspended in
medium containing 5% FBS and centrifuged. The cell pellets were washed twice in PBS
to remove any residual serum, and the washed cells were then plated at 106 cells /100 mm
plate or 50,000 cells/well of a 24 well plate in phenol red free DMEM F12 serum free

medium (Sigma) on collagen 1 (4 pg/cm2) coated tissue culture plates. After overnight

116

 

w.‘

incubation, cells were either treated with ICI 182,780 (100 nM), E (1 nM), IGF-l (10
ng/ml), ICI plus B, IGF-l plus B or IGF—l plus ICI. For ICI pretreatment, ICI was added

for 24 hr before IGF-l addition.

Plasmids. Heamagglutinin tagged AKT (pCEFL—myr-HA-AKT) contains a myristylation.
signal that causes membrane localization and has been described earlier (29). The
plasmid was a gift of Dr. Yi Li and J .S. Gutkind. The constitutively activate myc tagged
PI3K (pl 10*) and the kinase dead version (AKin) have been described previously (30),
and were provided by Dr. Anke Klippel. The SRE-luciferase and the CMV B-
galactosidase plasmid were obtained from Clontech. The cyclin D1 cDNA containing
plasmid that was used to prepare the probe for Northern Blotting was provided by Dr.
Steven Reed. A plasmid containing the 18S rRNA gene was obtained from Dr. Laura.

McCabe (Department of Physiology, Michigan State University).

Chemicals, Growth factor and Antibodies. ICI 182,780 was a gift of A. Wakeling
(Zeneca Pharmaceuticals) and l7B-estradiol was purchased from Sigma. IGF-l was
obtained from GroPep Pty. Ltd. (Adelaide, Australia) and the PI3K inhibitor, LY294002,
was obtained from Calbiochem. Rat tail collagen 1 was purchased from Collaborative
Biomedical Products (Bedford, MA). 3H-thyrnidine (50 Ci/mmol) was purchased from
ICN Biomedicals, Inc. Cyclin D1 antibody was a rabbit polyclonal from Upstate
Biotechnology. Antibodies to cyclin B, cyclin A and the pr protein were purchased .
from Pharmingen. ERor antibody was a monoclonal (Mabl7) antibody (31) and was a gift

of Dr. Richard Miksicek (Department of Physiology. Michigan State University).

117

 

Phosphoserine 473 AKT antibody and the AKT antibody were rabbit polyclonal
antibodies from New England Biolabs. Rat anti-HA and mouse anti-myc (clone 9E10.3)
antibodies were from Boehringer Mannheim and Neomarkers respectively. Sheep anti-
BrdU and mouse anti- BrdU were from Fitzgerald Industries and Boehringer Mannheim
respectively. FITC conjugated donkey anti-sheep antibody, goat anti-rat antibody were
from Sigma, while the rhodamine conjugated goat anti- mouse secondary was from

Boehringer Mannheim. The anti B-actin antibody was purchased from Sigma.

Transfections and Luciferase Assays. MCF-7 cells were plated at 5x105 cells/60mm
plate and transfected with plasmid DNA using the Superfect reagent following the
protocol provided by the manufacturer (Qiagen). 1 pg of SRE-luc plasmid was
cotransfected with either 1 pg vector, pl 10* or AKIN along with 0.2 pg of pCMV [3-
galactosidase as a control for transfection efficiency. Cells were kept in 5% charcoal
dextran stripped serum (CSS) containing medium for 24 hours followed by serum free
media for an additional 24 hours. Cells were harvested 48 h post-transfection and both
the luciferase and B—galactosidase activities were measured using the detection reagent
and protocol provided by the manufacturer (Clontech) on a Turner TD 20E luminometer
(Turner Designs). Each transfection was done in triplicate, and the luciferase activity in

each transfection was normalized to B-galactosidase activity.

3H Thymidine Incorporation. Cells were plated at 50,000 cells/well on collagen 1
coated 24 well plates and treated as described for the respective experiments. 1 pCi of

tritiated thymidine was added to each well and the cells were harvested in 0.5% NaOH,

118

0.1% Triton-X 100 after 24 hours of labeling. The cell lysates were precipitated with
trichloroacetic acid (10 %) and the acid precipitable counts were measured using liquid
scintillation counting. Six wells were harvested for each treatment with 3 wells being
used for thymidine incorporation and the remaining 3 wells used to determine the DNA
content by a fluorometric assay described previously (32). The 3H thymidine

incorporation per well was normalized to DNA content.

Immunoblotting. MCF-7 cells were lysed in Sherr lysis buffer (33) and the total protein
was quantitated using the Bradford protein assay (Biorad). Twenty micrograms of total
protein were subjected to SDS-Polyacrylamide gel electrophoresis (12% or 7.5%),
transferred to a PVDF membrane and probed for the various proteins using appropriate
primary antibodies followed by horseradish peroxidase conjugated secondary antibodies.
The bands were visualized using the ECL chemiluminescent reagent (Pierce). The cyclin
D1 levels on immunoblots for the time course experiment (Figure 4.2B) were quantitated

by densitometric scanning.

Northern Blotting. Cells were lysed using the Trizol reagent (GIBCO BRL) and total
RNA was extracted as described in the instructions provided by the manufacturer. Total
RNA was quantitated by measuring optical density at 260 nm, and 20 micrograms of
RNA was loaded on 1% formaldehyde gels, transferred to a nitrocellulose membrane and
hybridized with a 32P labeled cDNA probe for cyclin D1. The membrane was stripped

and reprobed with an 188 rRNA probe as a loading control. The specific RNA was

119

quantitated by phosphoimager scanning (Molecular Dynamics) and the cyclin D1 mRNA

levels were normalized to 18S rRNA.

Indirect Immunofluorescence. MCF-7 cells were plated on glass coverslips and
transiently transfected with either the HA-tagged AKT plasmid or the myc-tagged PIBK
(p110*) plasmid using the Superfect transfection reagent (Qiagen). After transfection,
cells were treated with medium containing 5% fetal bovine serum and ICI (100 nM) and
were labeled with 25 pM 5-Bromo-2’-deoxyuridine (BrdU) for 5 hours before fixation at
48 hours post transfection. The double immunofluorescence procedure for detecting
transfected proteins and BrdU has been described previously (34). BrdU incorporation
was detected using the appropriate or-BrdU antibody (mouse or-BrdU for HA-AKT and
sheep Ot-BrdU for myc—PI3K). Cells were viewed under a fluorescent microscope
(Olympus) and the percentage of HA (or myc) expressing cells that were also positive for
BrdU, and of cells that were positive for BrdU alone was determined. At least one
hundred HA/myc positive and negative cells were counted in each experiment. The
images shown in Figure 4.6A were taken on the Olympus microscope, scanned and

converted to grayscale using the Adobe Photoshop software program.

120

RESULTS

ICI decreases the proliferation of IGF-I treated cells To assess the effects of ICI, E
and IGF-1 on MCF-7 cell proliferation, cells were cultured in serum free medium (SFM)
and treated with ICI, E or IGF-l alone or with a combination of ICI plus B, IGF-l plus E
or IGF-l plus ICI. Untreated cells in SFM served as controls. Proliferation was assayed
by measuring 3H-thymidine incorporation over a 24 h labeling period beginning 24 hours
after plating. Results from one representative experiment are shown in Figure 4.1A.
MCF-7 cells in SFM showed basal levels of 3H thymidine incorporation that decreased 4-
5 fold upon treatment with ICI. This result suggested that a ligand independent activity of
ER was inhibited by ICI. The absence of E in the phenol red free SFM conditions was
confirmed by analyzing protein levels of ERa and progesterone receptor (Figure 4.1C
and data not shown). The levels of ERa were decreased in both ICI and E treated cells
compared to cells in SFM or treated with IGF-l. ICI has been previously shown to
decrease ERoc levels by causing receptor degradation (35), while E causes
downregulation of ERa by decreasing transcription and also by decreasing ERa mRNA
and protein stability of (36, 37). In addition, the levels of progesterone receptor protein, a
known transcriptional target of ER (38), were increased by E treatment of cells in SFM
(data not shown). These results confirmed that the concentration of E in SFM was below
the levels required to activate transcription from E target genes. E alone induced a 2-fold
increase in proliferation that was completely reversed by ICI. Treatment with IGF-l

alone increased proliferation 3-4 fold over control levels, and E had an additive effect

121

 

 

N
LII
CD

§

 

3H Thymidine incorporation 3>
cpm x 103/pg DNA '
G
o

100
50
0
SFMlCI ICI E - E ICI
+E +IGF-1
B.
.5 3x 4x
E A
o H
.92 4
§ '5 3
'5 E 2
E E
is 0
E SFM lGF-l ICI ICI
"1 +IGF-1
C.
5+ +IGF1

SFM ICI ICI E - E ICI

-.- -... cyclinDl

-- “...u w...“ cyclinE
--- a. on. «an. cyclinA
- . - “‘ ERa

. L : .. . .. .. ‘Actin

Figure 4.1. ICI decreases proliferation in the presence of IGF-1. MCF-7 cells were
plated in SFM, and after overnight culture were treated with ICI (100nM), E (lnM) or
IGF-l (10 ng/ml) or various combinations thereof as described in Materials and Methods.
Untreated cells in SFM served as controls. A) 3H thymidine (1 pCurie) was added to each
well after 24 hr treatment and cells were harvested after 24 hr labeling. Acid precipitable
counts were measured, and normalized to DNA content. Results from one experiment are
shown as the average +/- SE. from triplicate wells. B) 3H thymidine incorporation from
three independent experiments is shown as the average +/- SE. Incorporation is
expressed relative to the amount in SFM, which was defined as l. The fold induction of
proliferation by IGF—l over SFM or ICI treated cells is shown above the respective
histograms. C) In experiments carried out in parallel to the thymidine incorporation
studies shown in (A), cells were harvested after 2 days of treatment and lysates were
subjected to immunoblotting for cyclin D1, cyclin B, cyclin A and ERor. Actin served as a
loading control.

122

with IGF-l In agreement with published results (19, 20), ICI treatment decreased
proliferation of IGF-1 treated cells, but not to the level seen in cells treated with ICI
alone. In contrast, E induced proliferation was completely abrogated by ICI. We
determined the extent to which IGF-l stimulated proliferation in the presence and
absence of ICI and the results are shown in Figure 4.1B. Although the absolute levels of
proliferation were lower in ICI treated cells, IGF-l increased 3H thymidine incorporation

3-4 fold over basal levels in both untreated and ICI treated cells.

Both ICI and IGF-1 regulate cyclin D1 protein levels. To investigate the mechanism
by which ICI decreased proliferation, we examined the effects of ICI, E and IGF-1 on the
expression of cyclins, key regulators of the Gl-S phase transition. MCF-7 cells were
treated as described above, and after 2 days of treatment, protein extracts were prepared
and subjected to Western blot analysis for cyclins D1, E and A. Results from one
representative experiment are shown in Figure 4.1C. Cells in SFM expressed basal levels
of cyclin D1 that were decreased upon treatment with ICI. Treatment of cells with IGF-l
or with IGF-l plus E increased the levels of cyclin D1 compared to SFM controls.
Interestingly, cells co-treated with ICI and IGF-1 had lower levels of cyclin D1 than cells
treated with IGF-l alone. These results indicated that ICI decreased both basal and IGF-1
induced cyclin D1 protein levels. The levels of cyclin B protein did not show any changes
with the various treatments. Cyclin A protein is induced at the Gl—S transition, and serves
as a marker of S phase entry. Cells treated with ICI alone, or in combination with E or
IGF-l, showed decreased levels of cyclin A, correlating with the inhibition of

proliferation shown in Figure 4.1A and 4.1B.

123

ICI pretreatment does not abrogate IGF-l's ability to induce cyclin D1 expression
or proliferation. A number of studies have suggested that antiestrogens inhibit
proliferation by downregulating the IGF-l signaling pathway (24-27). Since in the
experiments described above, cells were treated simultaneously with both IGF-I and ICI,
it was possible that ICI had insufficient time to block IGF-l signaling and thus could not
inhibit its effect upon cyclin D1 expression and proliferation. To determine if ICI
pretreatment could abrogate IGF-l ’5 effects, cells were plated in SFM as described above
and were either untreated or preincubated with ICI for 24 hours. They were then
stimulated with IGF-l, harvested at various time points and analyzed by Western blotting
for cyclin D1, cyclin A, the Cdk inhibitory proteins, p21wan and p270“, and pr.
Results of one representative experiment are shown in Figure 4.2A. ICI pretreatment
decreased the levels of cyclin D1 compared to cells in SFM at the zero hour time point.
However, in both ICI pretreated and control cells; IGF-l induced cyclin D1 protein by 6
hours, with maximal levels being observed by 12 hours. The maximal levels of cyclin D1
expressed in ICI pretreated cells were approximately 2 fold lower than in cells treated
with IGF-l alone as determined by quantitation based on densitometric scanning. The
levels of cyclin D1 stayed constant in ICI plus IGF-l treated cells until 36 hours, but
showed a decline in cells treated with IGF-l alone between 24 and 36 hours. This
decrease correlated with the entry of cells into S phase, indicated by cyclin A induction
and pr phosphorylation. Since cyclin D1 protein levels vary in the different phases of
the cell cycle, with high levels in mid to late GI and decreased levels in S phase, the

decline in cyclin D1 levels is likely a consequence of cell cycle progression (6).

124

IC I+IGF—1 IGF- 1

 

0 612 24 36 0 612 2436 hrs
_ -, ,. cyclin D1

- . . .,

cyclin A

'0“

p27

“hum-1... “a“-.‘ww
Nu .. .. ....- u. .“ _,_’ w- 1.... h, p21

-_........ IIIII “fl“, Actin

. -1 1)pr
. M... .-.. m an 1:pr

”w“

B.
8
E < 12 3.4x
0 Z 10
912
8 no 8
.5 3
ca. 6
g Ti 4 2.4x
E E 2 ' '
>1 0
[5 0
: SFM IGF-l ICI ICI+

IGF—l

Figure 4. 2. ICI pre—treatment does not abrogate IGF-l induced expression of cyclin D1
or proliferation. MCF-7 cells incubated in SFM or pretreated with ICI for 24 hours were
stimulated with IGF-l. A) Cell lysates were harvested at the time points indicated and
subjected to western blotting for cyclin D1, cyclin A, p21wafi, p27k1", pr, or B-actin. B)
In a parallel experiment, 3H thymidine incorporation was determined during the 24 hr
period after IGF-l treatment. Results represent the average +/- SE. of triplicate wells
normalized to DNA content. Fold induction of 3H thymidine incorporation by IGF-l
treatment in either SFM or ICI treated cells is shown above the bars for the respective
treatments. The results shown are representative of three independent experiments.

125

In addition to decreasing cyclin D1 protein levels, ICI reportedly increases the

1Wafl

levels of the kinase inhibitors p27Kipl and p2 and these contribute to the inhibition of

proliferation (17). Since IGF-l decreases p27Kipl protein levels and may increase p21wa“
levels in MCF-7 cells (39), we assessed if ICI treatment could prevent the effects of IGF-
1 on these proteins. The p27KiP' protein levels were higher in ICI treated cells than
untreated cells at the 0 hr time point (Figure 4.2A). However, IGF—l treatment decreased

1wan were not

p27Kipl levels both in ICI pretreated and untreated cells. The levels of p2
reproducibly altered by IGF-l treatment in the presence or absence of ICI in these
experiments. These results indicated that ICI pretreatment could not prevent the effects of
IGF-I on both positive and negative regulators of proliferation, as exemplified by the
regulation of cyclin D1 and p27 Kip] protein levels.

The effects of ICI pretreatment on proliferation were also assessed. As shown in
Figure 4.2A, ICI pretreated cells showed small increases in cyclin A expression and pr
phosphorylation at the 36 hr time point. This was reflected in increases in levels of 3H

thymidine incorporation upon IGF-l treatment of ICI pretreated cells. Even in the

presence of ICI, IGF-l increased proliferation 2.4 fold over untreated cells (Figure 4.2B) .

Cyclin D1 mRNA and protein levels are coordinately regulated by ICI and IGF-1.

Mitogens can lead to the stabilization of cyclin D1 protein via AKT activation (28). Since
AKT is a downstream target of IGF-1 signaling, IGF-l treatment could lead to increases
in cyclin D1 protein without affecting cyclin D1 mRNA levels. If the levels of cyclin D1
mRNA induced by IGF-l in the presence and absence of ICI were similar, it would

support such a mechanism. To test this possibilty, total cellular RNA was extracted at

126

lCl +lGF-l lGF-l
0 4 8 0 4 8hrs

 

“" u U ‘5‘ cyclin D1

VJ -

a u w as» 1v ’4'" l8SrRNA

           

 

 

 

 

 

 

 

 

B. e
g omnmai
<5 Imm
ZS
‘E .72
5 .5
£8
5. {9' I I I
0 4 8
Time after lGF—l treatment (hours)
C- nmnmai
lle
A 3—
g S
B -:
“E :2 2
55 1-
E
5 E 0

0 4 8

Time after lGF-l treatment (hours)

Figure 4. 3. ICI pre-treatment does not prevent induction of cyclin D1 mRNA by IGF-l.
A) MCF-7 cells were incubated in SFM or SFM+ICI for 24 hours, and then stimulated
with IGF-l. At the times indicated after IGF-l treatment, total RNA was isolated and
analyzed for cyclin D1 mRNA by Northern blotting. B) Cyclin D1 mRNA levels were
normalized to 18$ rRNA and are represented as fold induction over the 0 hr ICI treated
time point. C) Cyclin D1 mRNA induction by IGF-l in the presence or absence of ICI is
represented as fold induction over the respective 0 hr time points. The results are the
mean +/- SE. of three independent experiments.

127

various time points after IGF—l stimulation of ICI pretreated or control cells, and
Northern blotting was carried out as described in Materials and Methods. Results from
one representative experiment are shown in Figure 4.3A and the quantitation of cyclin D1
mRN A induction in the presence and absence of ICI from three independent experiments
is shown in Figures 4.3B and 4.3C. IGF-l treatment increased cyclin D1 mRNA 2-3 fold
in both ICI treated and untreated cells. In Figure 4.3B, the amount of cyclin D1 mRNA at
the 0 hr time point in the presence of ICI was defined as 1 in each experiment, and other
values were expressed relative to this level. Consistent with the protein data, the absolute
levels of cyclin D1 mRNA attained are approximately 2 fold higher in the absence of ICI.
However as shown in Figure 4.3C, when the amount of mRNA at the 0 hr time point for
each treatment is defined as 1, the fold induction by IGF-l is similar (2-2.5 fold) in the
presence and absence of ICI. These results demonstrate that cyclin D1 mRNA levels
parallel protein levels in these experiments, and suggest that regulation of translation or
protein stability are unlikely mechanisms of cyclin D1 regulation by IGF-l. They also
demonstrate that both IGF-1 and ER signaling pathways regulate cyclin D1 mRNA

levels.

ICI treatment does not inhibit activation of AKT by IGF-l. It has been reported that
activation of PI3K by IGF-l is required to induce cyclin D1 protein and proliferation of
MCF-7 cells (4). We confirmed this requirement using LY 294002, a specific PI3K
inhibitor (40). Treatment with this inhibitor prevented the induction of cyclin D1 protein
(Figure 4.4A), mRNA and proliferation (data not shown) seen upon IGF-l treatment,

confirming a critical role for PI3K activity in IGF-l induced cyclin D1 expression and

128

IGFl IGF1+LY
0 6 12 6 12 hr

“- 4-“- ” nun-up mam-.4 ER“
* can. c—o ” ‘- " Actin
B.
ICI+IGF- 1 IGF- 1

--—-----+LY
I ll I

0 15 30 60 015 306030 min
* ' ..,........t “pAKT

 

..,”N-l

““h-ew“ ERa

-.... --- - .7 Actin

” vent-"V"
~ mu“ h.-

Figure 4.4. IGF-l induced PI3K activation is required for cyclin D1 expression, and is
inhibited by LY294002 but not by ICI. A) MCF-7 cells were incubated in SFM or
SFM+LY294002 (25 pM) for 30 min, treated with IGF-l (10 ng/ml) and lysates prepared
at the time points indicated. The lysates were analyzed by Western blotting for cyclin D1
and ERor protein. Actin was used as a loading control. B) MCF-7 cells were untreated,
pretreated with ICI for 24 hours, or pretreated with LY294002 (25 pM) for 30 minutes.
Cells were then stimulated with IGF-1, and lysates were prepared at the time points
indicated and analyzed by immunoblotting with an antibody specific for phosphorylated
Ser473 AKT. The blots were also probed with antibodies to total AKT, ERor and Actin.

129

proliferation of MCF-7 cells.

An important downstream target of PI3K is AKT (41). We reasoned that if ICI
inhibited IGF-l signaling upstream of PI3K, then it would prevent activation of AKT by
IGF-l. To test this possibility, MCF-7 cells were plated in SFM and incubated in the
presence or absence of ICI for 24 hours. IGF-l was then added and cell extracts were
prepared at various time points. Activation of AKT was assessed by immunoblotting with
a phosphospecific antibody that detects AKT phosphorylated on Ser 473 (42). The results
of one representative experiment are shown in Figure 4.4B. IGF-l treatment induced a
rapid phosphorylation of Ser473, with the levels of phosphorylated AKT being
maintained for 1 hour. This induction was not inhibited by ICI pretreatment, which
downregulated EROt protein levels. However, it was inhibited by treatment with the PI3K
inhibitor LY294002, indicating that induction of Ser473 phosphorylation by lGF-l does

not require EROt activity but does require PI3K activation.

Constitutively active PI3K or AKT does not confer ICI resistance. The above results
indicated that ICI does not inhibit the IGF-l signaling pathway leading to AKT
activation. Because PI3K is upstream of AKT, they also suggest that PI3K activity is not
regulated by ICI. To determine if activating PI3K could overcome the antiproliferative
effects of ICI, constitutively activated forms of a myc-tagged PI3K (p110*) or HA-
tagged AKT were transiently transfected into MCF-7 cells. The cells were labeled with
BrdU for 5 hr, then fixed and examined for AKT/PI3K expression as described in
Materials and Methods. As shown in Figure 4.5A, the tagged versions of both PI3K and
AKT were detectable in the cytoplasm and possibly the membrane of transfected cells.

Proliferation was assayed by determining the percentages of transfected. and

130

untransfected cells incorporating BrdU by double indirect immunoflourescence for BrdU
and either PI3K or AKT as described in Materials and Methods. The results indicate that
in the presence of ICI neither activated PI3K nor AKT was able to increase proliferation .
in the presence of ICI above the level seen in untransfected cells (Figure 4.5B).

Because constitutively active P13K (p110*) was unable to overcome an ICI
induced growth arrest, we confirmed the activity of this construct in MCF-7 cells by
assaying its ability to activate transcription from a serum response element (SRE)
promoter luciferase reporter. The SRE is a part of the complex promoters of genes such
as c-fos, and is activated by PIBK (43, 44). As shown in Figure 4.5C, the constitutively
active P13K (p110*), but not a kinase dead mutant (AKIN) of PI3K, induced a tenfold
increase in of SRE-luciferase promoter activity over vector alone, confirming that it is
active in transfected MCF-7 cells These results suggest that signaling via the PI3K

pathway is not sufficient to support proliferation of MCF-7 cells in the presence of ICI.

131

Figure. 4. 5. Constitutively active PI3K or AKT are insufficient to overcome an ICI
mediated cell cycle arrest. MCF-7 cells were transiently transfected with epitope tagged
constitutively activated AKT (HA-tag) or PI3K (myc-tag). After transfection, cells were
incubated for 48 hr in medium containing 5% FBS + ICI, and BrdU (25 pM) was added
for the final 5 hr before fixation. Cells in medium containing 5% FBS served as a control
population of cycling cells. Expression of the transfected proteins and BrdU
incorporation were detected by double indirect immunofluorescence as described in
Materials and Methods. A) Representative micrographs showing expression of the tagged
proteins. B) The percentage of PI3K or AKT expressing cells that were also BrdU
positive is represented by filled bars. Open bars represent the percentage of untransfected
cells that were BrdU positive in the same cultures. The results are the average +/- SE. of
three independent experiments. C) MCF—7 cells were transiently co-transfected with a
SRE-luc plasmid and either vector alone (V), constitutively active P13K (p110*) or a
kinase dead version of PI3K (AKIN). All transfections also included a CMV promoter
driven B-galactosidase plasmid as a control for transfection efficiency. The luciferase
activity was determined and normalized to B-galactosidase activity. The results represent
the fold luciferase induction over vector alone control, and are shown as the average +/-
SE. of three independent experiments, each done in triplicate.

132

01 HA (AKT) a-myc (PI3K)

 

B.
I HA+ormyc+

f”) 35 U HA- or myc-
: 30

.3 25

17. 20

8.15

D 10

'U

5 5

be 0

HA-AKT myc-PI3K FBS

C. FBS+ICI

916

.2

§12

d)

g 8

0

95 4

2

E 0

153 v AKin p110*

133

DISCUSSION

There is considerable evidence for cross talk between the IGF-1 and estrogen
signaling pathways in the regulation of breast cancer cell proliferation (45). Initial work
(19, 20) demonstrated that the antiestrogen tamoxifen inhibits insulin/IGF-l induced
proliferation of ER positive breast cancer cell lines, including MCF-7 cells. Results from
several laboratories have suggested that the decrease in IGF-l induced proliferation by
antiestrogens is the result of inhibition of the IGF-l signaling pathway, and that estrogens
and antiestrogens mediate their proliferative effects via this pathway. Both IGF-lR and
IRS-1 protein levels have been reported to be down regulated by ICI (25, 26, 46), as have
PI3K (27) and AKT (47) activities. Additional studies have indicated that E can enhance
both PI3K and AKT activation by IGF-l (39), and a recent study has reported rapid and
transient activation of P13K activity by E in MCF-7 cells (48). All of these findings
support the idea that estrogens and antiestrogens regulate the IGF-l signaling pathway.
However, evidence to the contrary also exists. Several studies have reported that E alone
does not affect either PI3K or AKT activity (39, 49). In addition, overexpression of IGF-
IR (50), IRS-1 (51) or constitutively active AKT (52) in MCF-7 cells does not lead to ICI
resistance, indicating that there are additional targets of ICI that mediate its anti-
proliferative effects.

In this report, we have investigated the effects of ICI on several targets of IGF-1
signaling. As previously reported, ICI treatment lowered the absolute level of
proliferation attained in IGF-l treated cells. However, ICI could not completely inhibit

proliferation in the presence of IGF-I. This can be explained by the fact that although ICI

134

treatment decreased basal proliferation in SFM, IGF-l could still induce proliferation (2-
4 fold) in the presence of ICI (Figures 4.1C and 4.2B). These results suggest that the
proliferative effects of IGF-I are not subject to inhibition by ICI. Similar results were
obtained with regard to the induction of cyclin D1, an important cell cycle regulatory
protein, by IGF-l. While the absolute levels attained were lower in the presence of ICI,'
the extent of both mRNA and protein induction by IGF-l were similar in the presence
and absence of ICI (Figures 4.2A and 4.3C). These results are in agreement with a recent
report (16) showing that ICI pretreatment of MCF-7 cells did not prevent cyclin D1
induction by insulin, which, at the concentrations used in that study, acts via the IGF-lR
(53). Similarly, IGF-l could down regulate the levels of p27K1’” in the presence of ICI,
demonstrating that ICI could not prevent the effects of IGF-1 on multiple cell cycle
components.

An interesting question raised by these results is whether the lower levels of
proliferation in IGF-l plus ICI treated cells relative to those treated with IGF-l alone is
due to lower cyclin D1 levels, or whether there are additional targets of ICI that
contribute to its effects. As shown in Figure 4.2B, although cyclin D1 is induced in the
presence of ICI, and accumulates to approximately 50% of the levels seen in the absence
of ICI, there is little hyperphosphorylated pr or cyclin A expression in the presence of
ICI. Since cyclin D1 can activate Cdk4 directly, and has been proposed to activate Cdk2
indirectly by titrating the Cdk inhibitor p21wan away from cyclin E-Cdk2 complexes into
cyclin Dl-Cdk4 complexes (13, 14), a certain threshold level of cyclin D1 expression
may be required to fulfill both of these functions. This possibility is supported by the fact

that a reduction in cyclin D1 levels by 50% using antisense oligonucleotides can result in

135

an almost complete inhibition of MCF-7 proliferation and cyclin E—Cdk2 activity (54).
However, E and ICI have also been reported to affect other cell cycle regulators including
c—myc, the Cdk inhibitors p21wan and p27K11’1, and the Cdk activating phosphatase
Cdc25A (13, 15, 17), and a combination of several of these effects is likely to be
responsible for ICI’s ability to decrease proliferation in the presence of IGF-I.

lGF-l activates several different intracellular pathways, including the MAPK and
PI3K signaling pathways (55, 56). Conflicting data has been presented regarding the role
of MAPK in E induced proliferation, with some studies showing no effects of E on
MAPK activity (57, 58) and others showing rapid and transient activation (48, 59).
Under our experimental conditions, MAPK activation by IGF-l was not regulated by ICI
treatment (data not shown). Numerous studies have indicated that the proliferative effects
of IGF-I in MCF-7 cells are mediated by PI3K. IGF-l treatment activates PI3K in MCF-
7 cells (3), and inhibition of PI3K activity with a specific chemical inhibitor LY 294002 I
(4), or by constitutive expression of PTEN, a phosphotidylinositol 3-phosphatase that
inhibits the PI3K pathway, causes a G1 arrest in these cells (60). To directly determine if
ICI inhibits or attenuates the PI3K pathway, we examined its effects on AKT activation.
AKT is implicated in both cyclin D1 induction and proliferation, and its phosphorylation
and activation are induced by PIBK (5, 61). Our results show that phosphorylation of
AKT on Ser 473 is induced by IGF-l in MCF-7 cells. This phosphorylation was inhibited
by LY 294002, but was unaffected by ICI treatment, indicating that the IGF-l signaling
pathway upstream of AKT is intact in ICI treated cells. Together with our findings that

neither constitutively active AKT nor P13K were able to promote proliferation in the

136

presence of ICI, these results establish that ICI does not block proliferation by inhibiting
the IGF— l/PI3K/AKT or the MAPK signaling pathways in MCF-7 cells.

There are several other possible mechanisms by which ICI treatment might lower
the absolute level of cyclin D1 expression and proliferation in the presence of IGF-I
including the following: 1) Both IGF-I and ER signaling may independently regulate cell
cycle components, and both may be necessary to promote full proliferation. 2) Part of the
effect of IGF-1 in MCF-7 cells may be due to its ability to activate the ER in a ligand
independent manner, and this effect would be neutralized in the presence of ICI since it
causes degradation of the ER. Additional work is required to distinguish between these
possibilities.

In summary, our results argue against a model in which ICI inhibits MCF-7 cell
proliferation by preventing the early signaling events induced by IGF—l including
PI3K/AKT or MAPK activities (See Figure 4.6 for model). Rather, they suggest that the
ER and IGF-1 signaling pathways converge to regulate cell cycle components including
cyclin D1 and p27m1’1 protein levels, and possibly others. Both pathways appear to be
required in order to obtain maximal proliferation, suggesting that deregulation of the
IGF-l signaling pathway in ER positive breast tumors would be insufficient to convert

them to an antiestrogen resistant phenotype.

137

IGF-1 E

LY ——l PI3K ER l——— ICI

./

Cyclin D1/Other cell cycle
components

1

pRB

Proliferation

Figure 4.6. Model of lGF-l and ER signaling interaction. IGF-l, by activating P13K
signaling regulates cyclin D1 and possibly other cell cycle components that lead to Cdk
activation. This in turn leads to pr phosphorylation and proliferation. Inhibition of the
PI3K pathway with a specific PI3K inhibitor, LY 294002, prevents the induction of
cyclin D1 and proliferation, indicating that this pathway is essential for IGF-l induced
proliferation. ER signaling does not interact with the IGF-l signaling via the PI3K
pathway but appears to converge on downstream cell cycle components including cyclin
D1. Treatment of cells with ICI does not inhibit IGF-l signaling. However, it decreases
pr phosphorylation and proliferation possibly by decreasing the absolute amount of
cyclin D1 induced and also by regulating other cell cycle components. According to this
model, activation of the IGF-l signaling intermediates would be insufficient to confer
antiestrogen resistance, whereas the inactivation of pr, which is downstream of IGF-1
and ER signaling, would result in antiestrogen resistance.

138

 

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CHAPTER 5

SUMMARY AND FUTURE EXPERIMENTS

A majority of breast cancers are estrogen dependent for proliferation making them
amenable to antiestrogen therapy. However, over the course of the disease, these tumors
progress to an antiestrogen resistant state resulting in treatment failure and contributing to
the high mortality associated with this disease. The focus of the studies described in this
thesis was to investigate the mechanisms by which breast cancer cells progress from an
antiestrogen sensitive to a resistant phenotype. The studies described have addressed two
distinct mechanisms that have been proposed to contibute to antiestrogen resistance.

Retinoblastoma protein (pr) is a negative regulator of cell cycle progression
whose inhibitory effect on proliferation is inhibited by phosphorylation by cyclin
dependent kinases (Cdks) Cdk4 and Cdk2. Estrogen treatment of MCF-7 cells, an
estrogen responsive human breast cancer cell line, leads to activation of Cdk4 and Cdk2
and increased pr phosphorylation. To establish whether pr is an important functional
target of estrogens and antiestrogens, we asked whether functional inactivation of pr
would allow MCF-7 cells to proliferate in the presence of antiestrogens. Using a panel of
SV40 and Polyoma virus large T antigens, we demonstrated that inactivation of pr is
required and may be sufficient for proliferation in the presence of antiestrogens. Further
experiments using inhibitors of Cdk4 and Cdk2 demonstrated that cells in which pr has
been inactivated still require Cdk2 activity for proliferation but no longer require Cdk4

activity. This suggests that Cdk4’s primary role in MCF-7 cells is to inactivate pr,

148

whereas Cdk2 has additional targets important for promoting proliferation. The facts that
antiestrogens inhibit Cdk2 activity and that Cdk2 activity is required for proliferation
upon pr inactivation in antiestrogen treated cells, suggests that Cdk2 activity must be
induced upon pr inactivation in the presence of antiestrogens. Further these results
suggest that estrogens regulate Cdk2 activity at least partially via a pr dependent
mechanism. Using stably transfected cell lines, we confirmed that expression of Polyoma
LT in ICI treated cells leads to Cdk2 activation. The mechanism of this activation has not
been characterized but it correlates with increases in cyclin A protein levels and may
therefore be due to formation of active cyclin A-Cdk2 complexes.

In summary, our experiments suggest a model for estrogen mediated cell cycle
progression, in which estrogen leads to Cdk4 activation, which then phosphorylates pr
and results in Cdk2 activation and proliferation of cells. Furthermore, the fact that pr
inactivation can promote proliferation in the presence of antiestrogens suggest that
inactivation of the pr pathway is a potential cause of antiestrogen resistance in breast
cancer patients. 1

Our experiments using viral T antigens also raised some interesting questions that
remain to be addressed. First, it is not known if pr inactivation by PyLT causes
activation of both cyclin B and/or cyclin A associated Cdk2. Since cyclin E-Cdk2 is
essential for progression to S phase, one would predict that its activity would be
increased upon PyLT induction. However, cyclin B levels are constitutive during G] in
MCF-7 cells. During estrogen-induced proliferation, increases in cyclin Dl-Cdk4
complexes have been proposed to sequester inhibitors from cyclin E-Cdk2 complexes,

thus activating cyclin E—Cdk2. Since we have not observed an increase in cyclin D1 or

149

cyclin E protein levels upon PyLT expression, the above cannot occur. It is also possible
that an increase in cyclin A-Cdk2 complexes leads to some inhibitor free active Cdk2
complexes. A second issue is that in stable cell lines, PyLT induced proliferation was two
fold lower than that seen in estrogen treated cells. This might be due to the fact that
approximately half the cells in the population did not express detectable PyLT. This
possibility can be tested by assaying BrdU incorporation in the presence of ICI in PyLT
positive and PyLT negative cells using double indirect immunofluorescence.
Additionally, in long term (8-12 days) assays, proliferation of PyLT expressing cells in
the presence of ICI was much lower than estrogen treated cells. The reason(s) for this are
unclear, but may involve one of several explanations. The inactivation of pr may not be
as efficient as estrogen treatment suggesting that estrogen has targets in addition to pr
that promote proliferation. If cyclin E-Cdk2 is not efficiently activated upon PyLT
expression, it would provide an explanation for decreased proliferation of PyLT
expressing cells in the presence of ICI in the long term. Finally, PyLT may be causing
growth inhibition or apoptosis in the long term, though in the short term this does not
appear to be the case.

The role of increased Insulin like growth factor-1 (IGF-l) signaling as a second.
potential mechanism of antiestrogen resistance was addressed in these studies.
Antiestrogens decrease proliferation of IGF-1 treated cells and it has been proposed that
they do so by downregulating IGF-l signaling. Our results suggest that this hypothesis is
not true. Cells treated with ICI did not show an inhibition of the IGF-l signaling
pathway, as determined by measuring activation of Akt or the induction of cyclin D] by

IGF-l. Furthermore, activation of either PI3K or Akt, intermediates in the IGF-l

150

signaling pathway that mediate IGF-l’s proliferative effects, was unable to induce
proliferation in the presence of ICI. Treatment of cells with ICI did cause a twofold
decrease in the absolute levels of cyclin D1 expression in IGF-l treated cells, and this
decrease correlated with decreased cyclin A protein levels, pr phosphorylation and
proliferation. These results suggest that both IGF-I and ER signaling are required to
promote proliferation in MCF-7 cells, and these two signaling pathways converge on cell
cycle components. They also predict that activation of the IGF-l signaling pathway alone

would be insufficient to confer antiestrogen resistance.

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