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This is to certify that the

dissertation entitled

ANTIESTROGEN RESISTANCE AND C1 PHASE CDK
REGULATION IN HUMAN BREAST CANCER CELLS

presented by
ANDREW JOHN SKILDUM

has been accepted towards fulﬁllment
of the requirements for

Ph.D. Microbiology
degree in

 

 

   

Major professor

July 29, 2002
Date

 

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Michigan State
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ANTIESTROGEN RESISTANCE AND G] PHASE CDK
REGULATION IN HUMAN BREAST CANCER CELLS

By

Andrew J. Skildum

A DISSERTATION

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

DOCTOR OF PHILOSOPHY
Department of Microbiology and Molecular Genetics

2002

ABSTRACT

ANTIESTROGEN RESISTANCE AND 6] PHASE CDK
REGULATION IN HUMAN BREAST CANCER CELLS

By

Andrew J. Skildum

Advances in detection and treatment have reduced the mortality of breast cancer
over the last decade, but it remains the second most lethal cancer for women in the US.
Antiestrogens, including Tamoxifen, have been successful therapies, but acquired
antiestrogen resistance during treatment remains a signiﬁcant barrier. A better
understanding of how estrogen and antiestrogen signaling controls proliferation in breast
cancer model systems may lead to the development of more efﬁcacious therapies which
avoid the problem of antiestrogen resistance. Estrogens and antiestrogens affect cell
proliferation at G1 phase, passage through which is governed by the sequential activation
of two cyclin dependent kinases, Cdk4 and Cdk2. In this report, we investigate the
mechanism(s) by which Cdk activity is regulated by estrogen and the steroidal
antiestrogen ICI 182780 in the MCF-7 cell line, a model of estrogen dependent,
antiestrogen sensitive human breast cancer. The hypothesis of this dissertation is that
defects in the regulation of Cdk4 and Cdk2 activities may be a cause of antiestrogen
resistant proliferation in in vitro models of human breast cancer.

Cdk activity is inhibited by antiestrogen in MCF-7 cells. Through analysis of
Cdk4 activity in estrogen and ICI treated MCF-7 cells by lysate mixing experiments, we
ﬁnd that extracts of ICI-treated cells contain a factor capable of inhibiting the Cdk4
activity present in extracts of estrogen-treated cells, and immunodepletion experiments

identify this factor as pZIWAFl/Cip'. We show that pZIW’WVCipl is an important ICI

mediated physiological regulator of Cdk4 which can inhibit its activity in the absence of
changes in levels of Cdk4’s main regulatory subunit, cyclin D1.

To examine the potential contribution of cyclin D1 overexpression to antiestrogen
resistance, we engineered a sub-clone of the MCF-7 cell line to conditionally express
epitope tagged cyclin D1 upon the addition of a synthetic drug. Cyclin D1
overexpression is sufﬁcient to allow cells arrested in G1 phase by antiestrogen to enter S
phase. Ectopic cyclin D1 activates Cdk4 in the presence of antiestrogen but does not
sustain long term Cdk4 activity or proliferation, and delays, but does not prevent,
antiestrogen mediated induction of the Cdk inhibitor p21“"‘mCipl and inhibition of Cdk4
activity in asynchronous cells.

Despite containing a normally functioning estrogen receptor, the MCF-7
derivative LCC9 cell line is able to proliferate in ICI and thus serves as a model of
acquired antiestrogen resistance. While in MCF-7 cells Cdk2 and Cdk4 are inactivated
by ICI treatment, we show that both kinases remain active in antiestrogen treated LCC9
cells. The Cdk4 from LCC9 cells retains sensitivity to inhibition by lysate from
antiestrogen treated MCF—7 cells. Compared to MCF-7, LCC9 cells have elevated levels

of cyclin D1 protein and unlike MCF-7 cells, p21wam “9'

is not highly regulated by
antiestrogen in LCC9 cells.

These experiments support our hypothesis that deregulated Cdk activity can
contribute to antiestrogen resistance, and suggest that altered signaling pathways

upstream of cyclin D1 and p21 may converge to maintain the activities of G1 Cdks in a

model of acquired antiestrogen resistance.

TABLE OF CONTENTS

LIST OF FIGURES .................................................................................. v
KEY TO ABBREVIATIONS ...................................................................... vi
CHAPTER 1: INTRODUCTION ................................................................. 1
Estrogen and breast cancer ................................................................. 3
Estrogen receptor signaling ................................................................ 3
Control of G1-)S phase transition ....................................................... 7
Antiestrogens .............................................................................. 12
Model systems used to study antiestrogen resistance ................................. 14
G1 checkpoint control in breast cancer cells ......................................... 18
Scope and signiﬁcance of this study ................................................... 20
References ................................................................................ 23

CHAPTER 2: REGULATION OF CDK4 BY ESTROGEN AND ANTIESTROGEN IN

MCF-7 CELLS .................................................................................. 38
Introduction .............................................................................. 40
Materials and Methods .................................................................. 43
Results .................................................................................... 46
Discussion ................................................................................. 61
References .................................................................................. 64

CHAPTER 3: INDUCTION OF CYCLIN D1 IN MCF-7 CELLS SUPPORTS SHORT

TERM PROLIFERATION IN THE PRESENCE OF ANTIESTROGEN ............... 69
Introduction .............................................................................. 71
Materials and Methods .................................................................. 74
Results .................................................................................... 77
Discussion .................... 88
References .......... 93

CHAPTER 4: CHARACTERIZATION OF THE LCC9 CELL LINE, AN

ANTIESTROGEN RESISTANT DERIVATIVE OF MCF-7 CELLS ..................... 97
Introduction .............................................................................. 99
Materials and Methods .................................................................. 102
Results .................................................................................... 106
Discussion .............................................................................. l 17
References .............................................................................. 121

CHAPTER 5: CONCLUSION ............................................................... 128
Introduction .......................................................................... 129
Signiﬁcance of major ﬁndings ......................................................... 130
Summary and model .................................................................. 136
References .............................................................................. l 3 8

iv

LIST OF FIGURES

CHAPTER 1
Figure 1: Schematic representation of estrogen mediated transcriptional activation...4
Figure 2: General model for progression through G1 phase of the cell cycle ........... 8
Figure 3: Structure of estrogen receptor ligands ........................................... 11

Figure 4: The development of in vitro models of acquired antiestrogen resistance 1 5

CHAPTER 2
Figure 1: Time course of Cdk4 inhibition by ICI in MCF-7 cells ....................... 47
Figure 2: Time course of Cdk4 activation by E2 in MCF-7 cells ....................... 49

Figure 3: Evidence for an ICI—regulated inhibitor of Cdk4 activity in MCF-7 cells ..51
Figure 4: Depletion of p21 but not p27 abolishes the Cdk4 inhibitory activity in ICI

treated MCF-7 cells ............................................................................ 54
Figure 5: Effects of p21 immunodepletion on the Cdk4 inhibitory activity in extracts of
ICI treated cells ................................................................................. 56
Figure 6: Cdk4 complex composition in E2 and ICI treated cells .......................... 59
CHAPTER 3
Figure 1: Construction of MCF-7-26-D cells .............................................. 78
Figure 2: Effects of cyclin D1 expression on growth arrested cells ..................... 80
Figure 3: Ectopic cyclin D1 activates Cdk4 in the presence of antiestrogen .......... 82
Figure 4: Expression of ectopic cyclin D1 delays, but does not prevent, inhibition of
Cdk4 and Cdk2 activity by antiestrogen .................................................... 84
Figure 5: Expression of exogenous cyclin D1 does not support long term proliferation
or Cdk4 activation in the presence of antiestrogen ........................................... 86
CHAPTER 4
Figure l: The LCC9 cell line's antiestrogen resistant phenotype is stable in the absence
of selective pressure ............................................................................ 107
Figure 2: LCC9 cells' estrogen receptor is regulated normally by E2 and ICI ........ 109
Figure 3: LCC9's G1 Cdks are not inactivated by antiestrogen .......................... 111
Figure 4: Cdk4 activity in LCC9 is sensitive to the inhibitory factor present in ICI
treated MCF-7 cells ............................................................................. 113
Figure 5: LCC9 cells have elevated cyclin D levels and deregulated p21 .............. 115
CHAPTER 5
Figure l: Alterations in regulation of G1 Cdks may lead to
antiestrogen resistance .......................................................................... 135

KEY TO ABBREVIATIONS

Abbreviations used are: AP, AP 1510. CAK, Cdk activating kinase. Cdk, cyclin
dependent kinase. Cde, Cdk inhibitor. CRE, CAMP response element. CSS, charcoal
stripped serum. DTT, dithiothreotol. EGF, epidermal growth factor. EGF-R, EGF
receptor. ER, estrogen receptor. ERE, ER response element. E2, l7B-estradiol. FBS,
fetal bovine serum. F KBP, FK506 binding protein. GST, glutathione-S-transferase.
Hours, h. ICI, ICI 182,780. IMEM, improved modiﬁed Eagle’s medium. IP,

immuno recipitation. Minutes, m. PKA, protein kinase A. PR, progesterone receptor.
p16, p16 K43. p21, pZIWAFl/C'p‘. p27, p27 “’1. Rb, retinoblastoma protein. SERM,
speciﬁc estrogen receptor modulator. 26D, MCF-7-26-D.

vi

CHAPTER ONE

Literature Review

Chapter 1: Literature Review

ABSTRACT

Breast cancer is a serious disease, annually accounting for over 1400 deaths in Michigan
and 40,000 deaths nationwide. Advances in detection and treatment have reduced
mortality over the last decade, but breast cancer remains the second most lethal cancer for
women in the US. Antiestrogens, including tamoxifen, have been successful therapies,
but acquired antiestrogen resistance during treatment remains a signiﬁcant barrier to
permanently curing breast cancer. A better understanding of the molecular biology of
signaling pathways that control proliferation in breast cancer model systems may lead to
more accurate diagnosis and the development of more efﬁcacious therapies. This
chapter reviews the mechanisms of estrogens’ action and their link to breast cancer,
antiestrogens and their limitations in breast cancer treatment, model systems used to
study estrogen signaling, and estrogen’s and antiestrogen’s effects on cell cycle
regulators. The conclusion provides an overview of the scope and signiﬁcance of the

research presented in the following chapters.

Estrogen and breast cancer

Estrogen has long been associated with the development and progression of breast
cancer (43). The most biologically active estrogen, 17B-estradiol (E2), is the primary
female sex hormone, driving neonatal development of the primary sex organs and further
differentiation in the genitals and breast during puberty. In addition, after puberty levels
of E2 ﬂuctuate over the 28 day menstrual cycle and during pregnancy. E2 is an organic
molecule derived from cholesterol; progesterone and testosterone are intermediates in E2
synthesis. E2 is produced primarily in the ovaries, though it is also produced by the
placenta during pregnancy, in the brain and in peripheral adipose tissue. In addition to
biological sources of E2, exposure to E2 and its analogs can also come from hormone
replacement therapy (HRT) and from organic pollutants and pesticides (15, 36, 111).

Although inheritance does play a role in the development of breast cancers in
some women, most cases occur sporadically. Estrogen is mitogenic in normal breast
tissue, and exposure to estrogen is considered to increase the risk of development of
breast cancer. Estrogen exposure may underlie other risk factors such as early puberty
and high fat diet. The greatest risk factor in the development of female breast cancer is
age, which can be considered the net sum of inherited, physiologic and environmental

risk and protective factors integrated over time (17, 85, 86).

Estrogen Receptor Signaling
E2 acts by binding to a nuclear steroid hormone receptor, the estrogen receptor

(ER). There are two isoforrns of ER, the well characterized ERa and the more recently

described ERB (106, 159). ERoc is a multidomain protein with hormone binding,
dimerization, and DNA binding domains, as well as two transcriptional activation
domains (AF-1 and AF-2). E2 binds the ligand binding domain and induces a
conformational change leading to receptor homodimerization and binding to DNA
upstream of E2 regulated genes at estrogen receptor response elements (EREs). ERa
then recruits co-activators via protein — protein interactions with the AF -1 and AF-2
domains (79, 87, 88, 91), stimulating transcription (Figure 1).

In addition to the classical mechanism described above, more recent
investigations suggest variations on this paradigm. ERa homodimers have been shown
to bind and activate promoters with non-consensus EREs (127). ERa and ERB also
regulate transcription at promoters containing AP-l sites by contacting Jun and F05
transcription factors and recruiting co-activators; this activity is independent of ER’s
DNA binding function. While E2 activates, and tamoxifen inhibits, ERa mediated
transcription at AP-l sites, ERB transcription at AP-l sites is inhibited by E2 and
activated by tamoxifen, and may account for cell type speciﬁc differences in the activities
of SERMs (92, 120, 166). Finally, ER splicing variants are often expressed in normal
and malignant breast tissue, and may serve as an additional level of regulation for ER
mediated transcription (83, 117).

ERor also can modulate transcription in the absence of ligand. Work by two
groups has shown that the cell cycle regulator cyclin D1 (discussed below) can bind ERa
and enhance transcriptional activation of EREs by forming a bridge between ER and

transcriptional co-activators (95, 110, 172, 173). The interaction between cyclin D1 and

A.

.l

I

 

C0-

 

 

 

EZ E2 activator,
@ ERa transcription
complex

  
  

 

I_E§E_J

[E2 regulated gene]

 

E2

 

co-
activator

 

 

 

      
 

transcription
complex

 

 

lEz regulated geneji

 

Figure 1: Schematic representation of estrogen mediated transcriptional activation.
Estrogen (E2) acts by binding to the estrogen receptor (ERa) in the nucleus. E2 binding
causes receptor dimerization. A) At E2 regulated promoters containing estrogen
response elements (ERE), ERa dimers bind to DNA and activate- transcription by
recruiting co-activators. B) At E2 regulated promoters which contain AP-l sites, ERa
binds jun and fos transcription factors without contacting DNA and recruits co-
activators, enhancing transcription. The ER isoforrn ERB has identical activity as ERa
at ERE containing promoters, but opposite activity at AP-l containing promoters: ERB
bound to E2 inhibits transcription of AP-l controlled genes.

ERoc occurs without Cdk4 activity or E2, although it is inhibited by antiestrogens and
inhibitors of cAMP signaling pathways. ERa is a substrate for Erkl and 2, mitogen
activated protein kinases (MAPKs), and phosphorylation inﬂuences the ligand
independent recruitment of transcriptional co-activators by AF-l (10, 46, 82, 158).

A major problem in the clinical management of breast cancer is the development
of antiestrogen resistance. E2 antagonists such as tamoxifen are used to successfully treat
ER positive breast cancer, but many patients’ tumors “evolve” to become resistant to the
drug; the resistant tumors are more aggressive and less treatable (108). Recent studies
have suggested that overexpression of the receptor tyrosine kinase Her2 and
phosphorylated, active MAPKs may correlate with relatively poor response to hormonal
therapy in ER positive tumors (54, 128). This suggests that overactive MAPK may cause
increased activation and ligand insensitivity of ERa, causing transcription and
proliferation in the presence of antiestrogen. MAPK overactivation may also cause
increases in non-ER regulated signaling pathways, bypassing blockade of ER by
antiestrogen and allowing antiestrogen insensitive growth.

These ﬁndings point to potentially novel ER signaling pathways in breast cancer
cells which may inﬂuence the outcome of hormonal therapy. Further examination of
these topics is necessary to determine which breast cancer cases are likely to develop
antiestrogen resistance and to develop alternative treatment modalities for patients with

this class of tumors.

Control of G1 95 phase transition

The effects of E2 and antiestrogens have been studied extensively in ER positive,
hormone responsive breast cancer cell lines, particularly the human breast
adenocarcinoma derived MCF-7 cell line. Early work showed that E2 acts to promote
progression through the G1 phase of the cell cycle, and that antiestrogens arrest sensitive
cells in G1 phase (93, 100, 116, 137, 151).

During G1 phase (G is for gap), normal mammalian cells biochemically assess
intra- and extracellular signals and “decide” whether or not conditions are appropriate to
proliferate. If conditions allow proliferation, cells pass a “restriction point”, aﬁer which
they are committed to enter S phase, when cells replicate their genomes. If a cell
perceives that conditions for growth are unfavorable, for example due to DNA damage or
a lack of a required hormonal context, the cell will arrest in G1 phase until conditions
improve. Non-transformed, differentiated cells may exit the cell cycle at G1 to enter G0,
a quiescent state. A hallmark of cancer cells is their lack of differentiation and
appropriate cell cycle control (39, 146).

The G1-)S phase transition in normal cells is controlled by two types of
serine/threonine kinases, cyclin dependent kinase-2 (Cdk2) and Cdk4/6. As their name
implies, catalytic activity of Cdks requires binding of a cyclin protein at one to one
stoichiometry. Cdk4, and the related Cdk6, bind to D type cyclins, including cyclins D1,
D2 and D3, although in most cell types, including most human breast cancer cells, cyclin
D1 is the major regulatory subunit of Cdk4. Cdk2 can bind either cyclin E or cyclin A.
Cyclins are proteins with short half lives whose levels peak at distinct points during the

cell cycle (12, 68, 69, 113, 121, 146, 154).

Mitogenic
signals

§

a
@9/ S hose
II 90-»@ ,5

\DNA replication
genes

 

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Figure 2: General model for progression through GI phase of the cell cycle. Many
mitogens activate signaling pathways that activate Cdk4 by increasing levels of
regulatory subunits such as cyclin D1 and/or decreasing levels of Cdk inhibitors such as
p21. Active Cdk4 phosphorylates Rb, which blocks its function as an inhibitor of E2F
family transcription factors. E2F induced genes include cyclins E and A, regulatory
subunits of Cdk2, and other genes involved in DNA synthesis. Cdk2 can phosphorylate
Rb, providing for ampliﬁcation of the mitogenic signal, as well as additional substrates
that promote the onset of genome duplication.

As shown schematically in Figure 2, active Cdk4 and Cdk2 can phosphorylate the
retinoblastoma protein (Rb). Rb is a member of the “pocket protein” family, containing a
cysteine rich pocket domain which binds numerous cellular proteins and viral antigens.

’ The Rb pocket also contains numerous Cdk2 and Cdk4 phosphorylation sites. While Rb
is the only known substrate for D-type cyclin binding kinases, Cdk2 also has additional
substrates directly involved in genome duplication, such as proteins in the origin
recognition complex (3, 80, 146).

In its unphosphorylated state, Rb binds E2F family transcription factors and
represses transcription of E2F regulated genes, resulting in growth arrest (61, 96); certain
viral proteins such as adenoviral ElA promote growth by binding to the Rb pocket and
displacing E2F (29). Similarly, after phosphorylation by Cdks, hyperphosphorylated Rb
no longer binds E2F, allowing transcription of genes that control S phase (167). The
expression of cyclins E and A is regulated by E2F, providing ampliﬁcation of the Rb
pathway’s signal (13, 60, 68, 72, 80, 102, 146). Certain mammalian cells, such as the
SAOS-2 osteosarcoma cell line, do not contain Rb, and no longer require Cdk4 activity
for proliferation (14, 63, 99) but still require Cdk2 activity (113).

In addition to cyclin binding, Cdk activity is also regulated by binding of small
proteins called Cdk inhibitors (Cdes). There are two families of Cdlds, represented by
p16ink4a (p16) and p21waﬂ/“p' (p21). p16, identiﬁed as a tumor suppressor protein, is a
speciﬁc inhibitor of cyclin D containing complexes, binding to Cdk4/6 and preventing
their association with an activating subunit (99, 144, 169). In contrast, p21, identiﬁed

independently as a Cdk interacting and p53 induced protein, forms ternary complexes

with all known Cdk/cyclin complexes (44, 45, 58); this ﬁmction is shared by the related
pz7kipl (p27) (130, 157).

Both p16 and p21 families have Cdk inhibitory activity, and both are reported to
block an activating phosphorylation of Cdk by Cdk-activating kinase (CAK) (7).
However, in certain experimental contexts, p21 and p27 may also serve as assembly
factors that enhance the formation and activity of cyclin D/Cdk4 complexes (30, 94, 168).
Whether p21 family Cdes serve as inhibitors or activators may depend on the
stoichiometry of their binding to cyclin/Cdk complexes, although there is some
controversy in this area (2, 59, 62, 109). Cde function may be regulated by other
mechanisms as well, such as proteolysis or phosphorylation (31, 70, 71, 142).

Based on numerous studies, a general model for the regulation of passage from
G1 to S phase in normal cells has been developed, and deregulation at various stages in
the model are proposed to contribute to cellular transformation and tumorigenesis (60, 68,
69, 98, 139, 146). Many mitogens increase cyclin D1 protein levels, either through
increased mRNA synthesis and/or stabilization of the protein. Cyclin D1 binds to
Cdk4/6, resulting in an increase in Cdk4/6 kinase activity, Rb phosphorylation and
synthesis of cyclins E and A. Increasing the cellular concentration of cyclin D1/Cdk4/6
complexes provides additional binding targets for p21 and/or p27, titrating these Cdes
from their inhibitory association with Cdk2 complexes (124, 129). Together, the
increased levels of cyclins and the decreased association of Cdes lead to Cdk2 activation
and progression to S phase (146). While this model is widely accepted, mitogens and
anti-mitogens can also affect progression through G1 in other ways, for example by

regulating the levels of Cdes (133, 135), the assembly and nuclear import of cyclin/Cdk

10

 

 

 

17B-estradiol

 

  

""(CH2)QSO(CH2)3CFZCF3

ICI 182,780

Figure 3: Structure of estrogen receptor ligands. Estrogen receptor ligands affect ER
transcription by binding to the ligand binding domain and inducing conformational
changes in the receptor, which result in modiﬁcations of ER's DNA and co-activator
binding behavior. A) The most biologically active estrogen, 17B-estradiol, is
representative of endogenous ER ligands. B) Tamoxifen is a triphenylethylene
antiestrogen that acts as a selective estrogen receptor modulator, having antagonist and
agonist activities in different tissues. C) The steroidal antiestrogen ICI 182,780 is a
pure estrogen receptor antagonist.

11

complexes (41), or the levels and activity of Cdk-activating kinases or phosphatases (51,

81,132).

Antiestrogens

Because of estrogen’s role as a mitogen in breast tissue, systemic estrogen
ablation has long been used for the treatment of breast cancer. Initially this was
accomplished surgically through ovariectomy (155). With the development of estrogen
antagonists (antiestrogens) in the 19605, E2’s effects were blocked chemically at the sites
of action in sensitive tissue. Antiestrogens bind ER’s ligand binding domain and inhibit
transcription, but individual compounds have their own unique properties. For this
reason antiestrogens are often referred to as selective estrogen receptor modulators
(SERMs) (75, 101). There are two broad classes, triphenylethylene antiestrogens and
steroidal antiestrogens (Figure 3).

The drug tamoxifen, a triphenylethylene compound, has been particularly
effective in the treatment of breast cancer when used as a supplement to surgery and/or
chemotherapy (143, 163). A current major problem in the treatment of breast cancer is
that patients who initially respond to Tamoxifen often develop tumors that have acquired
antiestrogen resistance and are independent of E2 (55, 76). Tamoxifen has also recently
been recommended for the prevention of breast cancer in women considered at high risk
for its development, without apparent concern for the potential implications on the
effectiveness of hormonal therapy if prevention fails (22).

In addition to concerns with resistance, Tamoxifen treatment causes a small

increase in the risk of uterine cancers; this may be due to its agonist activity in uterine

l2

tissue (6, 49). Agonist activity may also underlie the surprising ﬁnding that Tamoxifen
appears to mimic E2’s beneﬁcial effects on bone density (9, 141, 147). For these
reasons, other triphenylethylene antiestrogens have been developed which reduce the
undesirable side effects of Tamoxifen while maximizing the beneﬁcial effects of E2. For
example, Raloxifene, which has been shown to be as effective as Tamoxifen in breast
cancer prevention without increasing the risk for uterine cancers, also prevents
osteoporosis (40, 160). Continued efforts at developing antiestrogen compounds that are
only ER antagonists in the breast should improve treatment outcomes for breast cancer
patients.

Steroidal antiestrogens resemble E2 more closely than triphenylethylenes.
Steroidal antiestrogens have the same four ring cholesterol structure as E2, but with
carbohydrate substitutions at various positions. The drug ICI 182780 (ICI) is
representative of a class of steroidal antiestrogens which act as “pure” antiestrogens, with
no known agonist activity. Upon binding to ER’s ligand binding domain, ICI induces a
conformation in ER that does not allow interaction with co-activators and that causes
receptor degradation. Although acquired resistance to steroidal antiestrogens occurs
clinically and in in vitro models, early trials have shown ICI to be effective in breast
cancer patients who had previously failed triphenylmethylene treatment, suggesting that
resistance to steroidal and non-steroidal antiestrogens occurs by distinct mechanisms (38,
65,123)

Several mechanisms have been proposed to explain the acquisition of antiestrogen
resistance. These include a loss of ER or selection of ER negative cells from a

heterogeneous tumor population, a change in ERa or ERB function, altered metabolism

13

of antiestrogen, or inappropriate activation of estrogen dependent or independent growth
stimulatory signaling pathways; these alterations could occur alone or in combination and
could result from genetic lesions or epigenetic changes (33, 34, 64, 73, 122, 136). While
ER loss may be a cause of antiestrogen resistance prior to treatment, acquired resistance
during the course of treatment occurs in most cases without loss or mutation of ER (74),
and antiestrogen metabolism has not been shown to be a likely cause of antiestrogen
resistance (115). The composition of co-activators and/or co-repressors interacting with
antiestrogen bound ER may inﬂuence whether the ligand behaves as a transcriptional
activator or repressor at any given promoter, and changes in the makeup of ER containing
complexes may underlie the acquisition of antiestrogen resistance (57, 149, 153),
although clinical evidence has been lacking thus far (28). Much recent attention has
therefore been placed on alterations in signal transduction pathways regulating cellular

proliferation.

Model systems used to study antiestrogen resistance

Despite the importance of E2 and the widespread use of antiestrogens in breast
cancer treatment, the mechanisms by which these ER ligands affect proliferation in breast
tumor cells remain largely undeﬁned. A better understanding of ER signaling pathways
may lead to identiﬁcation of new therapeutic targets to improve breast cancer treatment.
Easily malleable model systems of E2 dependent, antiestrogen sensitive cells are required
to examine the molecular biology of ER signaling in breast cancer.

Most of our current understanding of E2 and antiestrogen action comes from in

vitro studies of cell lines derived from pleural effusions of patients with advanced

14

MCF-7

Estrogen dependent tumor formation LCC1

ICI/Tam sensitive in OVX nude mice ESt rogen Independent
ICI/Tam sensitive

 

 

selection in vitro selection in vitro selection in vitro
again st LY1 17018 against tamoxifen against ICI 182780
LY2 Estrogen independent Estrogen dependent
Estrogen independent Tam re$1$tant ICI/Tam resistant
Tam resistant ICI sensitive

Figure 4: The development of in vitro models of acquired antiestrogen resistance. The
MCF-7 cell line was derived from a metastatic breast adenocarcinoma. MCF-7 cells
require estrogen for proliferation and are inhibited by both triphenylethylene and
steroidal antiestrogens. Through selection of MCF-7 cells for tumorigenicity in
ovariectomized nude mice, the estrogen independent but antiestrogen sensitive LCCl
cell line was derived. Compared to MCF-7, LCCl cells have increased expression of
matrix metalloproteinases and nucleophosmin. The LCC2 cell line was developed by
selection of LCCl cells for growth in tamoxifen in vitro. LCC2 cells are resistant to
triphenylethylenes, but remain sensitive to steroidal antiestrogens, and have increased
ratio of anti-apoptotic Bcl-2 to the pro-apoptotic Bax, and have increased expression of
TGFB. LCC9 cells were selected from LCCl cells by selection for growth in ICI
182780, and are resistant to both types of antiestrogens; aside from their growth
phenotype LCC9 cells remain largely uncharacterized.

Independently, LY2 cells were selected directly from MCF-7 cells for resistance to a
Raloxifene analog in vitro. LY2 cells show hyperactive MAPK, leading to
phosphorylation and degradation of the Cde p27.

15

metastatic breast cancer. Well characterized ER positive human breast cancer cell lines
include ZR-75-1 (4, 47) and T47D (145), but by far the most widely studied is MCF-7.
The MCF-7 cell line was derived from a 69 year old Caucasian female with metastatic
breast adenocarcinoma who had failed previous radiation and hormone therapy (21, 150),
although the cell line is hormone sensitive.

In order to study the clinically relevant acquisition of antiestrogen resistance,
investigators have attempted to recreate the phenomena in vitro (Figure 4). Through
selection of MCF-7 cells for estrogen independent tumorigenicity in ovariectomized
nude mice, Clarke and colleagues derived the LCC] cell line (23, 32), which is
independent of estrogen for growth but retains sensitivity to antiestrogens. Further
selection of LCCl in vitro with increasing concentrations of Tamoxifen yielded LCC2,
which is estrogen independent, resistant to Tamoxifen yet sensitive to growth inhibition
by steroidal antiestrogens (25, 35), supporting the clinical observation that patients who
had failed Tamoxifen treatment were still effectively treated by ICI. Selection of LCC]
in vitro with increasing concentrations of ICI led to the LCC9 cell line, which is resistant
to ICI and to triphenylethylenes, despite not being exposed to them during the selection
process (24).

Together, these four cell lines represent a model of stepwise acquired antiestrogen
resistance in human breast cancer. As is often the case with clinical antiestrogen
resistance, ER expression is unchanged in the LCC series of cell lines (24, 25, 156).
Likewise, antiestrogen metabolism is unchanged during the acquisition of antiestrogen
resistance (77). This suggests that other mechanisms besides loss of ER or altered drug

metabolism are responsible for the growth phenotypes of these cell lines.

16

Compared with MCF-7 cells, estrogen independent LCCl cells show increased
metastatic potential, correlating with increased matrix metalloproteinase expression
(138). LCCl cells also have higher levels of nucleophosmin, an inhibitor of interferon
regulatory factor-1 (IRF -1), a tumor suppressing transcription factor involved in growth
arrest (90, 148), which may contribute to an increased growth potential in the absence of
hormone signaling.

The tamoxifen resistant LCC2 cells are reported to have increased levels of TGF-
[3, a peptide hormone that inhibits the proliferation of normal breast tissue and early stage
breast cancer cells, but is often overexpressed and mitogenic in advanced breast cancer
(8, 140). LCC2 cells have also been shown to have elevated Bel-2 and decreased Bax
expression (97). The pro-apoptotic Bax forms homodimers and heterodimers with Bel-2,
and when Bcl-2 - Bax dimers predominate, apoptosis is inhibited in cells (114).
Tamoxifen is reported to cause death of breast cancer cells by apoptosis (26, 119), and
the LCC2 cell line’s elevated Bel-2 to Bax ratio may increase their growth potential in
tamoxifen by making them less sensitive to normal apoptotic pathways. LCC2 cells are
also reported to have increased levels of an ERa splice variant, and altered nuclear matrix
protein organization compared to their tamoxifen sensitive progenitors (8, 89, 97, 131),
although the possible role of these changes in LCC2 cells’ tamoxifen resistant phenotype
is not clear. As of the preparation of this manuscript, no reports describe potential
mechanisms for the phenotype of the steroidal antiestrogen resistant LCC9 cell line.

In contrast to the stepwise selection used to generate the LCC model of acquired
antiestrogen resistance, another antiestrogen resistant derivative of MCF-7, the LY2 cell

line, was selected directly for in vitro resistance to a Raloxifene analog (20). LY2 cells

17

retain wild type ER yet, compared to MCF-7, LY2 show deregulated E2 mediated
expression of the transcription factor P82, but not the progesterone receptor (PR) (37,
107). This cell line has been shown to have hyperactive MAPK signaling leading to
hyperphosphorylation and degradation of p27 a, and treatment with speciﬁc MAPK-
kinase inhibitors restores antiestrogen sensitivity (42).

It is apparent that multiple changes accompany the development of estrogen
independence and antiestrogen resistance in the above models of human breast cancer.
However, the precise molecular determinants of antiestrogen resistance remain
undeﬁned. Further study of these and novel models of breast cancer progression may
reveal commonalities that will suggest new therapeutic strategies that avoid or delay the

development of antiestrogen resistance.

GI checkpoint regulation in breast cancer cells

The important roles of cyclins and Cdkls in regulating cell proliferation prompted
investigation into their expression in primary breast tumors and correlation with other
parameters, such as clinical outcomes and ER expression. Approximately 60% of breast
tumors are ER positive, and although ER expression correlates with a more benign tumor
grade, endocrine therapy achieves complete remission in only one half of patients with
ER positive cancer (1, 50, 56, 118).

Cyclin D1 protein is overexpressed in one third of breast cancers, and in a third of
these cases, the cyclin D1 gene, CCNDl, located on chromosome 11q13, is ampliﬁed
(48, 53, 78, 126, 171). Cyclin D1 ampliﬁcation and protein overexpression correlate

positively with ERa expression in breast cancer (48, 66, 152, 161 , 171). Although cyclin

18

D1 overexpression has been consistently reported in primary tumors, reports of its
prognostic signiﬁcance vary considerably. The overexpression of cyclin D1 has been
reported to improve relapse free survival (18, 125), to be predictive of poor prognosis if
in combination with positive immunostaining for ER, epidermal growth factor, or Rb (84,
104), and to have no correlation with tumor grade or prognosis (152, 161). These results
suggest that in breast tumors, other factors inﬂuence the effects of cyclin D1
overexpression on the course of disease.

In addition to cyclin D1, Cdkls have been investigated in primary breast tumors.
Although oﬁen mutated in other cancers, p16 mutation appears to be infrequent in breast
cancer, although changes in p16 expression have been reported due to either homozygous
deletion or alterations in promoter methylation, and overexpression of pl 6 is associated
with increased tumor malignancy (16, 19, 67, 105). p21 mutation appears infrequently in
breast tumors as well, although one point mutated p21 gene that encodes a mutant p21
capable of binding, but not inhibiting, Cdk complexes was isolated from primary breast
tumors (1 l, 103). Positive staining for p21 correlates with ER and p53 expression, lower
tumor grade, and improved clinical outcomes in breast cancer patients (112, 162). These
results suggest that in primary breast cancer, maintenance of mechanisms which normally
regulate the activities of Cdk4 and Cdk2 is linked to maintenance of ER and to less
advanced disease.

Based on these observations, E2 and antiestrogens’ effects on Cdk regulation of
the G1 -)S phase transition were investigated further using in vitro models of hormone
sensitive breast cancer such as the MCF-7 cell line. Like many cancer derived cell lines,

the MCF-7 cell line has inherent defects in cell cycle regulation, including deletion of

19

p16 and constitutively expressed cyclin E (170), however other aspects of E2’s mitogenic
inﬂuence on MCF-7’s cell cycle machinery resemble normal cells (Figure 2).

Others have reported that antiestrogen treatment of MCF-7 cells decreases cyclin
D1 and cyclin A protein levels, Cdk4 and Cdk2 activity and Rb phosphorylation, and
increases Cde levels (27, 52, 129, 164, 165). All of these effects are reversed upon
removal of antiestrogen and replacement with estrogen (5, 51, 134). Although both total
Cdk2 and cyclin E/Cdk2 activities are highly regulated, cyclin B protein is expressed at
constant levels in estrogen and antiestrogen treated MCF-7 cells. The lack of activity of
cyclin E/Cdk2 complexes in the presence of antiestrogen is believed to be due to
inhibition by p21 and/or p27 in the complex. Cyclin E/Cdk2 activation after estrogen
treatment may therefore be a result of decreased levels of these inhibitors and/or
sequestration of p21 or p27 into newly formed cyclin D1/Cdk4 complexes (129, 134).

These observations suggest that E2 and antiestrogens inﬂuence proliferation by
signaling through ER to modulate Cdk activity through multiple mechanisms. Disruption
of one or more of these mechanisms may account for the acquisition of antiestrogen
resistance in breast cancer cells. Further study to address the precise function of cyclins
and Cdkls in ER mediated proliferation, as well as to deﬁne ER’s signaling pathways

upstream of Cdks, is warranted.

Scope and significance of this study
Acquired antiestrogen resistance is a major problem in the management of breast
cancer, and antiestrogens exert their antiproliferative effects in sensitive cells by blocking

the activation of G1 phase Cdks. The hypothesis of this Dissertation is that alterations in

20

the regulation of Cdk4 and/or Cdk2 activities are responsible for acquired resistance to
the steroidal antiestrogen ICI in in vitro models of human breast cancer. Work described
in the subsequent chapters addresses this hypothesis using three approaches:

1) We attempted to identify key mediators of antiestrogen’s antiproliferative
effects in the ICI sensitive MCF-7 human breast cancer cell line (Chapter 2). We found
that Cdk4 activity can be regulated by E2 and ICI independently of cyclin D levels, and
that the Cde p21 is responsible for inhibiting Cdk4 after ICI treatment.

2) MCF-7 cells were engineered to conditionally express cyclin D1, a component
of the Rb pathway implicated in ER signaling, and the effects on hormonal responses
were tested (Chapter 3). Short term ICI resistance was achieved by overexpressing cyclin
D1 in MCF-7 cells; ectopic cyclin D1 activated and co-precipitated with endogenous
Cdk4. Cyclin D1 delayed, but did not prevent, ICI’s upregulation of p21, and cyclin D1
overexpression was not sufﬁcient to allow long term proliferation in ICI.

3) The composition and kinase activities of Cdk4 and Cdk2 complexes observed
after E2 and ICI treatment of MCF-7 cells were compared with those observed in an ICI
resistant MCF-7 derivative, LCC9 (Chapter 4). While Cdk4 and Cdk2 activities were
inhibited by ICI in MCF-7 cells, in LCC9 the Cdks remain active, although LCC9’s Cdk4
remained sensitive to inhibition by p21. LCC9 cells express higher levels of cyclin D
than MCF-7, and their p21 is not upregulated by ICI.

Based on the results of these studies, we propose that the proliferative potential of
hormone sensitive breast cancer cells depends on a positive factor, cyclin D1 , and a
negative factor, p21, the balance of which determine the activity of G1 Cdks (see model

Chapter 5, Figure 1). Expression of ectopic cyclin D1 leads to Cdk4 activation and

21

proliferation in the presence of antiestrogen, and antiestrogen resistant LCC9 cells have
overexpressed cyclin D1 and unregulated p21, although the general function of their Rb
pathway appears intact. The hypothesis that disruption of the Rb pathway can cause
antiestrogen resistance is thus accepted, with the qualiﬁcation that in our model of
acquired antiestrogen resistance, the disruption(s) appears to be caused by factors
upstream of cyclin D1 and p21 expression rather than in cyclin D1 and p21 themselves.
Future studies will examine the control of cyclin D1 and p21 expression by ER ligands to

assess its impact on the development of antiestrogen resistance.

22

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37

CHAPTER TWO

Regulation of Cdk4 by estrogen and antiestrogen in MCF-7 Cells

Originally published as Skildum, A. J., S. Mukherjee, and S. E. Conrad. 2001. The cyclin
dependent kinase inhibitor p21WAF” C'“ is an antiestrogen regulated inhibitor of Cdk4 in
human breast cancer cells. J Biol Chem 277(7):5145-52.

38

Chapter 2: Regulation of Cdk4 by estrogen and antiestrogen in MCF—7 Cells

ABSTRACT

The MCF-7 cell line is a model of estrogen dependent, antiestrogen sensitive
human breast cancer. Antiestrogen treatment of MCF-7 cells causes dramatic decreases
in both Cdk4 and Cdk2 activities, which leads to a G1 phase cell cycle arrest. In this
report, we investigate the mechanism(s) by which Cdk4 activity is regulated in MCF-7
cells. Through time course analysis, we demonstrate that changes in Cdk4 activity in
response to estrogen or antiestrogen treatment do not correlate directly with cyclin D1
protein levels or association. In contrast, Cdk4 activity does correlate with changes in the
level of the Cdk inhibitor p21WAF ” Cipl. Furthermore we show that extracts of
antiestrogen-treated cells contain a factor capable of inhibiting the Cdk4 activity present
in extracts of estrogen-treated cells, and immunodepletion experiments identify this
factor as p21WAH/C’p'. These results identify p21‘MF”Cipl as an important physiological

regulator of Cdk4 complexes in human breast cancer cells.

39

Introduction

Many estrogen receptor (ER)I positive breast tumors require estrogen for growth,
and can be successfully treated with antiestrogens (40, 41). The MCF-7 cell line, which
was derived from a human breast adenocarcinoma, serves as a model for such estrogen
responsive, antiestrogen sensitive, breast tumors (22). MCF-7 cells require estrogen to
proliferate, and arrest in the G1 phase of the cell cycle when they are deprived of
estrogen or treated with antiestrogens (24, 27, 28, 45). Understanding the mechanisms by
which antiestrogens arrest the growth of breast cancer cells is an area of active
investigation with potential clinical signiﬁcance.

In normal cells, the transition from G1 to S phase requires the activity of two
classes of cyclin dependent kinases (Cdks), Cdk4/6, and Cdk2. Cdk4/6 phosphorylate the
tumor suppressor protein pr, which leads to transcription of E2F- regulated genes
including cyclins E and A. Cdk2 can also phosphorylate pr, as well as additional
substrates necessary for genome duplication (13, 14, 42, 43). Cdk activity is regulated by
multiple mechanisms, including phosphorylation (9, 19, 33, 52) and association with both
positive and negative regulatory proteins, and each of these mechanisms is potentially
modiﬁed by ER signaling. Cdk activation requires association with a cyclin partner:
Cdk4/6 associate with D type cyclins, while Cdk2 associates with either cyclin E or
cyclin A (7, 16). Cdk activity can be inhibited by two different families of cyclin
dependent kinase inhibitors (Cdkls). Members of the INK4a family, including p16INK4a

(p16), bind speciﬁcally to monomeric Cdk4/6 and prevent its association with a D type

 

' Abbreviations used are: ER, estrogen receptor. p21, WAFl/Cipl. p27, Kipl. p16, INK4a. Cdk, cyclin
dependent kinase. Cdkl, Cdk inhibitor. pr, retinoblastoma protein. 152, 17B-estradiol. IP,
immunoprecipitation. 1C1, 1C1 182,780. DTT, dithiothreotol. IMEM, improved modiﬁed Eagle’s medium.
CSS, charcoal stripped serum. Minutes, m. Hours, h. FBS, fetal bovine serum.

40

cyclin (30, 37). Members of the WAF l/Cipl family, which include p21WAF”Cipl (p21)
and p27kipl (p27), bind to G1 cyclin/Cdk complexes, not to monomeric cyclins or Cdks
(11, 17, 20, 30, 32, 46, 47). While both p21 and p27 can inhibit the activity of Cdk2 and
Cdk4/6, p21 can also serve as an assembly factor for cyclinD/Cdk4 complexes, increasing
the efﬁciency of complex formation and Cdk4 activity (5, 12, 21).

Based on numerous studies, a general model for the regulation of passage from
G1 to S phase in normal cells has been proposed, and deregulation at various stages in the
model are proposed to be responsible for cellular transformation and tumorigenesis (14,
16, 17, 23, 39, 42). Many mitogens induce cyclin D expression, either through
increased mRN A synthesis and/or stabilization of the protein. Cyclin D binds to Cdk4/6,
resulting in an increase in Cdk4/6 kinase activity, pr phosphorylation and synthesis of
cyclins E and A. Increasing the cellular concentration of cyclin D/Cdk4/6 complexes
provides additional binding targets for p21 and/or p27, thereby titrating these Cdkls from
their inhibitory association with Cdk2 complexes (30, 31). Together, the increased levels
of cyclins and the decreased association of Cdkls lead to Cdk2 activation and progression
to S phase (42). While this model is widely accepted, mitogens and anti-mitogens can
also affect progression through G1 in other ways, for example by regulating the levels of
Cdkls (34, 38), the assembly and nuclear import of cyclin/Cdk complexes (6), or the
levels and activity of Cdk-activating kinases or phosphatases (9, 19, 33).

The speciﬁc mechanism(s) by which estrogen and antiestrogens regulate MCF-7
cell proliferation are not completely understood. Others have reported that antiestrogen
treatment decreases cyclin D1 and cyclin A protein levels, Cdk4 and Cdk2 activity, and

pr phosphorylation, and increase Cde levels (4, 10, 31, 48, 49). All of these effects are

41

reversed upon removal of antiestrogen and replacement with estrogen (2, 10, 36).
Although both total Cdk2 and cyclin E/Cdk2 activities are highly regulated, cyclin B
protein is expressed at constant levels in estrogen and antiestrogen treated MCF-7 cells.
The lack of activity of cyclin E/Cdk2 complexes in the presence of antiestrogen is
believed to be due to inhibition by p21 and/or p27 (31, 36). Cyclin E/Cdk2 activation
after estrogen treatment may therefore be a result of decreased levels of these inhibitors
and/or sequestration of p21 or p27 into newly formed cyclin Dl/Cdk4 complexes (31,
36).

Previous reports suggest that Cdk4 activity in estrogen or antiestrogen treated
MCF-7 cells is regulated primarily by the levels of cyclin D1 protein. Although p21 and
p27 are found in association with Cdk4, evidence for a physiological role in inhibiting
Cdk4 is lacking. The Cdkl p21 is reported to be both an activator and an inhibitor of
Cdk4, so its function in the regulation of Cdk4 activity by antiestrogen is unclear. In the
current report, we investigate the mechanisms by which l7B—estradiol (E2) and the pure
antiestrogen ICI 182,780 (ICI) regulate Cdk4 activity in MCF-7 cells. We ﬁnd that Cdk4
activity is not directly correlated with cyclin D1 protein levels or association. Utilizing
an in vitro mixing assay, we demonstrate that extracts of ICI-treated cells contain a factor
that inhibits the Cdk4 activity present in extracts of estrogen treated cells, and that this
inhibitory factor is speciﬁcally removed by immunodepletion of p21 but not p27. These
studies identify p21 as an important physiological target of antiestrogen action that

inhibits both Cdk4 and Cdk2 activities in human breast cancer cells.

42

Materials and Methods

Cell lines and culture media. MCF-7 cells were obtained from Dr. Michael Johnson of
the Lombardi Cancer Center, Georgetown University. They were routinely passaged in
IMEM (BioFluids), supplemented with 5% fetal bovine serum (FBS, HyClone), 100
units/mL penicillin (Gibco) and 100 ug/mL streptomycin (Gibco). For experiments, cells
were cultured in IMEM without phenol red (BioFluids) containing 5% charcoal stripped
serum (CSS, HyClone) and penicillin / streptomycin, with either 10'8 M E2 (Sigma) or

10'8 M ICI (Astra Zeneca). Cells were cultured at 37° C with 5% C02.

Cell cycle analysis. Cells were trypsinized, washed in phosphate buffered saline (PBS),
suspended in PBS+10% F BS, ﬁxed with 80% cold ethanol and stored at —20° 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-lOO. Cells were then
analyzed for red ﬂuorescence on a F ACSVantage ﬂow cytometer; cell cycle distribution
was determined using ModFit software. Three 60 mm plates were analyzed for each time

point. These experiments were performed in part by Shibani Mukherjee.

Immunoprecipitations and Cdk4 kinase assays. Immunoprecipitations (IP) and Cdk4
activity measurements were performed using a modiﬁcation of a published method (25).
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

43

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, lmM NaVO4, 0.1 mM PMSF, 10 ug/ml leupeptin, and 2 [lg/{1’11 aprotinin). The
lysates were cleared by centrifugation, and protein concentrations were quantitated using
BioRad’s protein assay reagent. For IPs, 75 pg of total protein was diluted to 500 111 with
IP 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) bound to 7.5 111 protein G agarose beads
(Boehringer Mannheim). Beads were pelleted and washed four times with 100 pl IP
buffer and twice with 100 1.11 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 40 111 kinase buffer containing 10 pCi y-[32P]ATP, 20 11M
cold ATP, and 1.0 111 glutathione sepharose 48 (Amersham Pharrnacia) to which
approximately 2.0 pg of a fusion protein between glutathione-S-transferase (GST) and
amino acids 792 to 928 of human Rb were bound (GST-Rb bacterial expression vector
was kindly provided by Dr. William Kaelin of the Dana F arber Cancer Institute). 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 polyacrylamide gels, which were then dried and exposed to autoradiography
ﬁlm. The data were quantitated by phosphorimaging with a Storm phosphorimager

(Molecular Dynamics) using ImageQuant software.

44

Western blotting. Either total cell lysates (20 pg), IP supematants or IP pellets were
resolved on 12% SDS-polyacrylamide gels, transferred to membranes and probed with
antibodies for cyclin D1 (UB1 06 137, Upstate Biotechnology), p21wan (p21-c-19, Santa
Cruz Biotechnology), p27k’p1 (C-19, Santa Cruz Biotechnology), Cdk4 (H22, Santa Cruz
Biotechnology) or actin (Sigma clone AC-40). Membranes were then incubated with
horseradish peroxidase conjugated goat anti-rabbit (BioRad) or goat anti-mouse
(American Qualex) secondary antibodies, and immunoreactive proteins were detected

using Super Signal West Pico Chemiluminescent Substrate (Pierce).

Lysate mixing assays. Cells were harvested, stored, and lysed as described above.
Seventy ﬁve pg of protein from E2 treated cells were mixed with 7.5 to 75 pg of protein
from ICI treated cells, and diluted to 500 p1 with IP buffer. The mixtures were incubated
at 30° C for 30 m with occasional agitation. The mixed lysates were then subjected to the
Cdk4 kinase assay as described above. Phosphorylated substrate band intensities were

quantitated by storage phosphorimaging.

Immunodepletion. For immunodepletion studies, 300 pg of lysates were diluted to 600 p1
with IP buffer and incubated rocking at 4° C with 22.5 pl protein G agarose beads to
which was bound 4.5 pg (Figures 4 and 5) or 9 pg (Figure 6) antibody (p21-C-19-G, p27-
C-19-G, Cdk4-H22-G or normal goat IgG, all from Santa Cruz). The beads were

pelleted, and the supernatant incubated with fresh antibody bound beads a second time.

Aliquots of the supematants and pellets were then analyzed for Cdk4 activity, by Western

45

blotting or used in the lysate mixing assay as described above. Statistical analysis for

data presented in Figure 5 was performed using Microsoft Excel.

Results

ICI and E2 regulate Cdk4 activity independent of cyclin D] levels or association in
MCF-7 cells. Cyclin D/Cdk4 complexes are key integrators of positive and negative
growth signals (16, 17, 23, 42, 44), and both cyclin D1 levels and Cdk4 activity are
regulated by E2 and ICI in MCF-7 cells (10, 31, 36). To determine whether changes in
cyclin D1 protein levels and/or association correlate directly with Cdk4 activity and cell
cycle progression in these cells, we investigated the effects of ICI and E2 on cell cycle
distribution, Cdk4 activity and cyclin D/Cdk4 complex formation. The experiments were
performed in the presence of charcoal stripped serum (CSS), which is free of steroid
hormones.

To study the inhibition of proliferation by ICI, MCF-7 cells were plated at low
density and incubated in the presence of E2 for 48 h to generate an asynchronous,
proliferating population. The E2 containing medium was then removed and fresh
medium containing ICI was added to block ER signaling, and cells were harvested at six
hour intervals. As shown in Figure 1A, ICI caused a gradual decrease in the percentage
of cells in S phase beginning by 18 h after treatment, and reaching a minimum of 5% by
48 h. Under these conditions, Cdk4 activity was reduced 50% by 12 h, and 90% by 24 h
(Figure 1B). The decrease in Cdk4 kinase activity preceded the observed changes in cell
cycle phase distribution, consistent with the concept that the loss of Cdk4 activity was

responsible for the cell cycle arrest observed.

46

% Cells in S Phase
1?"? C,’ ”7 E ”7 1°:
_
I
i!
1'
I

0h 6h 12h 18h‘24h 30h‘36h 48h
Time After ICI Treatment

E
B. g
Hours after E Cdk4 | P
lCItreatment: 0 0 6 12 24 3o
GST-Rb —> m “ ’ .»
‘é
C 6
' Hours after lysates g Cdk4 |p

 

'0 treatment: 0 612 24 3O 0 0 612 24 30

m- ‘_ Cdk4

W- ‘_ ACtln

Figure 1: Time course of Cdk4 inhibition by ICI in MCF-7 cells. MCF-7 cells
were cultured in medium containing 10-8 M E2 for 48 h, washed in PBS, then
treated with medium conatining 10-8 M ICI and harvested at the times indicated.
A) Cells were ﬁxed, stained with propidium iodide, and analyzed by ﬂow cytometry
as described in Materials and Methods. The average percentage of cells in S phase
at each time point is shown +/- one stande deviation (n = 3). B) Cells were
treated as in IA and assayed for Cdk4 activity as described in Materials and
Methods. Numbers indicate hours aﬁer ICI treatment. The lane labeled mock IP
shows kinase activity precipitating with pre-immune control antibodies. C) Lysates
and aliquots of the reaction from (B) were irnmunoblotted for cyclin D1, Cdk4, p21,
p27 and actin. Data shown are representative of three independent experiments.

47

To determine if the decrease in Cdk4 activity shown in Figure 1B correlated with
changes in cyclin D1 and/or Cdkl protein levels, Western blot analyses were performed
(Figure 1C). No changes in Cdk4 protein levels were detected, conﬁrming that the
decrease in Cdk4 activity was not due to a decrease in the level of the catalytic subunit.
These analyses also indicate that Cdk4 activity did not correlate directly with cyclin D1
protein levels. At 24 and 30 h after antiestrogen treatment, when there was virtually
complete inhibition of Cdk4 activity, the cyclin D1 levels in the lysates were similar to
those observed in asynchronous MCF-7 cells prior to antiestrogen treatment (0 h). More
signiﬁcantly, the amount of cyclin D1 co-immunoprecipitating with Cdk4 did not change
decrease detectably during the time course. While the levels of p27 remained relatively
constant during this experiment, the levels of p21 increased aﬁer ICI treatment, and this
increase correlated with Cdk4 inactivation (Figure 1C). These data indicate that the
inhibition of Cdk4 activity following antiestrogen treatment is independent of changes in
cyclin D1 levels, and suggest that increased p21 levels may inhibit Cdk4.

To further examine the effects of ICI and E2 on Cdk4 activity, the above
experiment was performed in reverse. Cells were pre-arrested with ICI, released by the
removal of ICI and the addition of E2, and harvested at 6 h intervals. At each time point,
samples were analyzed for cell cycle distribution, for Cdk4 levels and activity and for
cyclin D1, p21 and p27 protein levels and Cdk4 association. As shown in Figure 2A, the
percentage of cells in S phase remained low until 18 h, then increased to 40—50% at the
24 and 30 h time points. Cdk4 activity was undetectable at 0 h, and increased by 24 and
30 h (Figure 2B). In agreement with the results presented in Figure 1, activation of Cdk4

did not correlate with changes in the levels of cyclin D1 or in its association with Cdk4.

48

 

 

 

 

 

 

 

50
0)
v1 Eh
g 40i .f
“ I "'1‘ 1:1
2 3° 1 ; Li 1
a 20 ?‘
u i 3
59 10 1 ; :3
0 WELL-fhﬂﬁ
011 611 12h 18h 24h 3011
Time After 152 Treatment
E
x
3
Hours after Cdk4 l P E
Eltreatment o 6 12 18 24 30 30
“" "" ‘— GST-Rb
&
1?
Hours after U 5315 Cdk4 11’ E
E2treatment: 0 6 12 18 24 3O 0 6 12 18 24 30 30
I-”""""“** CyclinDI "- ---...__

.""""-H p27 'D..-. .....

~~~~¢~ ACtln

Figure 2: Time course of Cdk4 activation by E2 in MCF-7 cells. MCF-7 cells
were arrested for 48 h in medium containing10-8 M ICI, washed, treated with
medium containing 10-8 M E2 and harvested at the time points indicated. A)
Cell cycle analysis was performed as in Figure 2A. B) Samples were assayed for
Cdk4 activity as described in Materials and Methods. C) Aliquots of total cell
lysates and of the immunoprecipitates from (B) were Western blotted for cyclin
D1, Cdk4, p21, p27 and actin. This experiment has been repeated once with
similar results (not shown).

49

A transient increase in cyclin D1 was reproducibly observed at 6 and 12 h after treatment
in both the total lysate and the Cdk4 IP, but this increase was not coincident with Cdk4
activation. When Cdk4 activity was highest, at 24 and 30 h after E2 treatment, cyclin D1
protein levels were similar to before treatment (Figure 2C). In this experiment, both p21
and p27 levels decreased after E2 treatment in both the total cellular lysate and Cdk4 IP.
These data, taken together with the results of Figure 1, suggest that Cdk4 activity in E2 or
ICI treated cells is not directly determined by the total amount of cyclin D1 in the cell or
by the amount of cyclin D1 in complex with Cdk4. Both experiments provide correlative
data suggesting that p21 and/or p27 might be responsible for the inhibition of Cdk4

activity in response to ICI treatment.

ICI regulates Cdk4 activity through an inhibitory factor. The results presented above
provide correlative data that accumulation of p21 and/or p27 might be responsible for the
inhibition of Cdk4 activity in these ICI treated cells. We therefore designed in vitro
mixing experiments to test this possibility directly. MCF-7 cells were pre-arrested in ICI
for 48 h, then treated with medium containing either E2 or fresh ICI for an additional 24
h. Lysates were prepared, and increasing amounts of lysate from ICI treated cells were
mixed with a constant 75 pg of lysate from E2 treated cells. The single and mixed
lysates were diluted to a constant volume, incubated for 30 m, and then subjected to a
Cdk4 activity assay as described in Materials and Methods.

This experiment was designed to distinguish between three potential mechanisms
for regulating Cdk4. If the lack of Cdk4 activity in ICI-treated cells was due to a post-

translational modiﬁcation of the complex, such as a change in phosphorylation state, we

50

 

 

2"le Cdk4
IP: Cdk4 mock Cdk4 1:1
_ _ _ 1:10
2 3 LJ 3 LJ 8 L“.
Q Qle 111111 a + GST-Rb

B. a b c A B c

a? ’ i

> E

E i

m .

E

..0.5

0

C

.9

6 El

3 o vvvvv

110 15121 ICI
C. lysate c_dk42 IP mocklP
ICI E2 10 £2 ICI £2

Cyclin DI —> ‘ II— ..... ..

p2] -> - " -...

Actin—> - .

Figure 3: Evidence for an ICI-regulated inhibitor of Cdk4 activity in MCF-7 cells.
MCF-7 cells were pre-arrested with ICI for 48 h, then treated for 24 h with either ICI or
E2. A) Samples were assayed for Cdk4 activity as described in Materials and Methods.
Supematants from samples marked with lower case letters were subjected to a second
round of Cdk4 IP and kinase assay, shown in lanes with the corresponding upper case
letters. For mixes, 75 pg of E2 treated lysate were added to increasing amounts of ICI
treated lysate in a constant volume of 500 ml. Unmixed reactions were diluted to 500
p1, and all tubes were incubated at 30° C for 30 In prior to immunoprecipitation for
Cdk4 assays. B) Three independent mixing experiments were performed as described
in (A) and quantitated by phosphorimaging. For each experiment, the amount of
activity in the E2 treated extract was deﬁned as 1, and activities recovered in the mixed
extracts are expressed relative to that value. The average activities are shown +/—
standard deviation is shown (open bars) compared to the predicted activity expected
based on the ratio of active to total lysate in the mixtures (closed bars). C) Lysates and
immunoprecipitates from (A) were immunoblotted for cyclin D1 , p21 and actin.

51

would expect the Cdk4 activity in the lysate of E2-treated cells to be unaffected by the
addition of the lysate from ICI-treated cells. If the lack of Cdk4 activity was due to the
absence of a Cdk4 activator (such as a D type cyclin), we might expect to see an increase
in total activity in the mixed lysates. Finally, if the lack of Cdk4 activity was due to the
presence an inhibitory factor, we would expect the addition of the extract of ICI-treated
cells to lower the activity present in the E2 treated lysates.

The results of one representative mixing experiment are shown in Figure 3. As
predicted from the time course experiments described above, only background levels of
Cdk4 activity were recovered from the ICI treated cells, and a 10 fold increase in Cdk4
activity was observed after E2 treatment (Figure 3A, lanes 1 and 2). Under these
experimental conditions, the amount of immunoprecipitating antibody was limiting, so
that the Cdk4 activity recovered was not the total activity in the lysate but rather a
representative fraction thereof. This is demonstrated by the fact that equal activity was
detected in a second sequential Cdk4 IP of the same lysate (Figure 3A, lanes 5-7, marked
“2“d 11)”).

Because the IPS were performed in “lysate excess”, we predicted that the mixed
lysates would exhibit decreased Cdk4 activity in the absence of any activating or
inhibitory activities. Since Cdk4 is present at equal levels in active and inactive extracts
and IPs (see Western blots in Figure 2C; note the constant level of Cdk4 detected in Cdk4
IPs despite differences in kinase activity), and since the mixed reactions contained a
greater amount of total cellular protein, the predicted decrease would be proportional to
the ratio of inactive to active lysate in the mixture. For example, at the 1 to 2 mixing

ratio, 37.5 pg of ICI treated lysate was mixed with 75 pg of E2 treated lysate, and the

52

predicted Cdk4 activity in the mixture would be 75/(75 + 37.5) = 0.67 = 67% of the
activity of the E2 treated lysate; this fraction is represented by black bars in Figure 3B.

Three identical mixing experiments were performed, and the activity recovered in
mixed lysates was expressed as a fraction of the activity recovered from the E2 treated
lysate. As shown in Figure 3B, at each mixing ratio the amount of activity recovered was
signiﬁcantly less than the fractional representation of the E2 treated lysate in the
mixtures. These results provide the ﬁrst direct evidence that ICI treatment induces a
factor capable of inhibiting Cdk4 activity, and that this factor is present in excess of
cyclin D/Cdk4 complexes in ICI treated lysates.

Aliquots of the lysates and Cdk4 IP pellets used in Figure 4A were analyzed for
cyclin D1 and p21 protein levels by Western blotting (Figure 3C). Consistent with our
previous result, similar amounts of cyclin D1 were present in the lysates from ICI and E2
treated cells, despite the differences in kinase activity. The amount of p21 in both the
total lysate and in complex with Cdk4 was slightly higher in the ICI-treated than in the

E2-treated lysate.

Depletion of p21 , but not p2 7, removes the Cdk4 inhibitory activity from ICI treated
lysates. To determine whether a known protein accounted for the inhibition of Cdk4
activity in extracts of ICI-treated cells, we modiﬁed the lysate mixing experiment
described above. Prior to mixing, aliquots of the ICI treated lysate were subjected to two
rounds of immunodepletion with antibodies against p21, p27, Cdk4, or with a normal
goat IgG control (mock). The immunodepleted ICI treated lysates were then incubated

with E2 treated lysate at a 1:1 mixing ratio and subjected to a Cdk4 kinase as described

53

A' ICI 52
ID: - p21 p27Cdk4 m -

 

. ' E 1' 1-
i Hi. I"...
v ‘- , k ‘7‘ ' .

, A “'4:- J4—p27

““ﬂ *- -~ In»- <-—Cdk4

- .--.- <—Cyclin DI

 

 

 

 

 

 

 

 

 

 

 

<—Actin
B mocklP
'0 E2f‘j Mix: |C|_+E2
V .5: st .2
1— Nx U PK .2 U
'0 '3382'- -aaag
" '- - - -<—GST-Rb
C.
31.2 -- ,1 _ - ,- -- _, --_.,_____j
.210 2
E ' ’7
790.8—
30.6— __
o
E 0.4“
60.2 *
gooi— D ' D 1:1 ,2:
52 E2+ICI 10
ID: - - p21 p27 Cdk4 mock -

Figure 4: Depletion of p21 but not p27 abolishes the Cdk4 inhibitory activity in [CI
treated MCF-7 cells. The Cdk4 inhibition assay was carried out as described in Figure
4, but with the ICI treated lysate subjected to two rounds of immunodepletion with anti-
p21, anti-p27, anti-Cdk4 or control (goat IgG, "mock") antibodies prior to mixing with
the E2 treated lysate. A) Aliquots of the untreated and immunodepleted lysates were
immunoblotted for p21, p27, Cdk4, cyclin D1 and actin. Lanes marked " - " contain
untreated lysates. M, mock immunodepletion. B) Autoradiogram of the Cdk4 in vitro
kinase assay. The ﬁrst seven lanes show the Cdk4 activity in unmixed lysates; the last
ﬁve lanes show Cdk4 activity in lysates mixed at 1:1 ratios. C) Bands in (B) were
quantitated by phosphorimaging. The Cdk4 activities in the mixed lysates (E2 + ICI) are
expressed as a fraction of the activity in the E2 treated lysate. The dashed line represents
50% of the activity in the lysate from E2 treated cells, which is the fractional
representation of the E2 treated lysate in the mixtures. This experiment was repeated
once with similar results (not shown).

54

above. Aliquots of the untreated and depleted extracts were also analyzed by Western
blotting for p21, p27, Cdk4, cyclin D1 and actin levels.

As shown in Figure 4A, two rounds of immunodepletion were sufﬁcient to
signiﬁcantly reduce the amount of the target proteins in the ICI treated lysate, although
all were detectable by Western blotting with very long exposures. Two rounds of
treatment with a control antibody (goat IgG, “mock”) had no effect on the levels of the
proteins examined. Immunodepletion of either Cdk4 or p21 removed the majority of
cyclin D1 from the lysate of ICI treated cells, suggesting that virtually all the cyclin D1 in
1C1 treated cells is in complex with both Cdk4 and p21. Immunodepletion of Cdk4
reduced the amount of p2 1 , while immunodepletion of p21 resulted in only a slight
decrease in the amount of Cdk4 present in lysates from ICI treated cells. Depletion of
p27 did not change the levels of p21, Cdk4 or cyclin D1, although we have consistently
observed p27 co-precipitating with Cdk4 (see Figure 6), as reported by others (5, 46).

Figure 4B shows the autoradiogram of the in vitro kinase assay for this
experiment; the quantitation of phosphorylated GST-Rb band intensities is shown in
Figure 4C. Immunodepletion of p21 or p27 had no effect on the Cdk4 activity recovered
from the ICI treated lysate, indicating that the removal of inhibitors alone is not sufﬁcient
to activate Cdk4. Consistent with the results described in Figure 3, mixing the non-
immunodepleted ICI treated lysate with the E2 treated lysate resulted in inhibition of
Cdk4 activity to less than the 50% predicted by the fractional representation of the E2
treated lysate in the mixture. When the ICI treated lysate was immunodepleted of p27,
Cdk4 or treated with control antibodies, a similar inhibition of the Cdk4 activity in the E2

treated lysate was observed. However, immunodepletion of p21 from the 1C1 treated

55

 

 

 

    

    

 

 

O.
A. .3
supematants Cdk4 IP 2
ID: - p21 mock- - p21mock- -
Q Q Q a Q Q Q m a
p21-y . - - . _
Actin-> "~ -- «- u...
m
B. E
U
E NolD 21 ID mocle
”3‘ ‘ P21 "1°C": ' 15 1:1 1': 1:1 15 1:1
9 Q Q a a /l /I /I
“ H I? It at at up w .<-GST—Rb
C.
1.2 “"
Z‘ 1
IE 1.0 l
‘0' 1
_‘_° 0.8 1
N F
‘5 1
3: 0.6
O
C
.3 0.4 1
1
L“ 0-2 g“ l
\‘ !
E2 1:5 1:2 1 1 ICI

Figure 5: Eﬂects of p21 immunodepletion on the Cdk4 inhibitory activity in extracts
of ICI treated cells. Mixing assays were carried out as described in Figure 4. Prior to
mixing, aliquots of ICI treated extract were subjected to two rounds of
immunodepletion with anti-p21 or control antibodies (mock). A) Aliquots of IP
supematants and IP pellets were immunoblotted for p21 and actin. B) Representative
autoradiogram of a Cdk4 kinase assay. Lanes marked " - " were undepleted. C)
Three replicate experiments were quantitated by phosphorimaging, and the fractional
activity recovered from the mixed lysates relative to the activity in the E2 treated
lysate was plotted as described in Figure 4. Gray bars, Cdk4 activity from undepleted
extracts from ICI treated cells and mixtures thereof. Hatched bars, Cdk4 activity in
mock depleted ICI treated extracts and mixtures. White bars, Cdk4 activity from p21
immunodepleted extracts and mixtures. Black bars, fractional representation of the
E2 treated lysate in the mixed lysates. Error bars represent one standard deviation.

56

lysate resulted in a loss of inhibition; the amount of Cdk4 activity recovered in the
mixture was approximately 60% of the amount in unmixed E2 treated lysate (Figure 4B),
which is greater than the 50% predicted if no inhibitor was present. We therefore
conclude that the Cdk4 inhibitory activity present in ICI-treated MCF-7 cells is the Cdkl
p21.

To conﬁrm the role of p21 in regulating Cdk4 activity, additional
immunodepletion/mixing experiments were performed. MCF-7 cells were pre-arrested
with ICI, treated with ICI or E2, and lysates were then prepared as described above. The
ICI—treated lysate was immunodepleted of p21 and mixed with the E2 treated lysate at
three different mixing ratios. The mixed lysates were then subjected to Cdk4 in vitro
kinase assays. Three independent experiments were performed, and the average
inhibition observed in the mixed lysates was compared to the predicted value based on
the proportional representation of the E2 treated lysate as described above. Aliquots of
both the lysates and the Cdk4 immunoprecipitates were also analyzed by Western
blotting for p21 and actin.

As shown in Figure 5A, two rounds of immunodepletion qualitatively removed
p21 from both the total lysate and from Cdk4 immunoprecipitates, while treatment with
control antibodies (goat IgG, “mock”) had no effect on p21 levels. A representative
autoradiogram from these experiments is shown in Figure 5B, and the average results
from the three independent experiments are shown in Figure 5C. Consistent with the
results in Figure 4, removal of p21 from the ICI treated lysate abolished the Cdk4
inhibitory activity. At each mixing ratio, the Cdk4 activity recovered was signiﬁcantly

higher when p21 depleted ICI-treated lysate was mixed with E2 treated lysate than when

57

non-depleted or IgG-depleted lysates were used. A one tailed t-test was performed to
compare the Cdk4 activities recovered from mixtures of undepleted lysate with mixtures
of IgG depleted lysates (51). The p values were 0.31, 0.14, and 0.19 for the 1 to 5, 1 to 2
and 1 to 1 mixing ratios respectively. When undepleted mixtures were compared with
p21 depleted mixtures, p values were 0.015, 0.023 and 0.020. When IgG treated mixtures
were compared with p21 depleted mixtures, p values were 0.017, 0.011 and 0.0013.
These data conﬁrm an important role for p21 in inhibiting Cdk4 kinase activity in

antiestrogen treated human breast cancer cells.

The majority of cyclin D1 in E2 treated MCF—7 cells is in complex with both p21 and
Cdk4. Since cyclin D1 was co-immunodepleted with both Cdk4 and p21 antibodies from
extracts of ICI treated cells, we conclude that virtually all of the cyclin D1 was present in
complexes containing both Cdk4 and p21 under these conditions (Figure 4A). To
investigate whether a pool of p21-free cyclin D1/Cdk4 complexes accounted for the
Cdk4 activity in extracts of E2-treated cells, additional immunodepletion experiments
were performed on extracts of both E2 and ICI-treated cells using antibodies directed
against p21, p27 and Cdk4.

As shown in Figure 6A, cyclin D1 was almost completely removed by
immunodepletion of either Cdk4 or p21 in extracts of both E2 and ICI treated cells. This
conﬁrms that the majority of cyclin D1 in ICI treated MCF-7 cells is in complex with
both Cdk4 and p21, and demonstrates that this is also true in E2 treated cells. Thus, even
though p21 levels are decreased by E2 treatment, the p21 remaining is sufficient to co-

immunoprecipitate the majority of the cyclin D1. Immunodepletion of p27 partially

58

 

 

 

 

 

supematants . Pellets
d
ID: - p21 p27 Cdk4 mock ”fund 21mm"
_ ._ _ — — _ LU _. LU
232328128213 ( Cdk4“- .2...
M .~ 4-Cdk4 . .-
-- . a... Cyclin Dl .
-- {-Cyclin DI . ' .
Cdk4 < 921 «1......
'1‘ M.w.‘.+p21 p27 “-uw
mm-‘ n “ +1327 K ,p27 he - 2...
M +actin mock 927 ‘m ‘m g ‘4

Cdk4 “- ~

p21 Cyclin DI ’- 7".

p21 --

1327 Cyclin D1 ~ -- .

Figure 6: Cdk4 complex composition in E2 and ICI treated cells. Cells were arrested
with ICI for 48 h, then treated for 24 h with either ICI or E2. Extracts were prepared and
immunodepleted of p21, p27 or Cdk4 as described in Figure 4, with the exception that the
amount of antibody used in each depletion was doubled. Mock-depleted extracts were
treated with preimmune goat IgG. A) Cell lysates and supematants from the second
immunoprecipitation were analyzed by Western blotting for levels of Cdk4, cyclin D1,
p21, p27 and actin. B) Pellets from the sequential immunodepletions were analyzed by
Western blotting for Cdk4, cyclin D1, p21 and p27. This experiment was repeated once
with similar results. The Cdk4 IP, p27 Western blot marked with an asterisk is from the
second experiment.

59

removed cyclin D1 from both lysates (Figure 6A), and immunodepletion of Cdk4
lowered p27 levels. Since most of the cyclin D1 is removed by immunodepletion of p2 1 ,
this suggests that at least a subset of cyclin Dl/Cdk4 complexes must contain both p21
and p27.

Others have suggested that there is a redistribution of p21 and/or p27 from Cdk2
to Cdk4 complexes after E2 treatment (31, 36). As shown in Figure 6A, there appears to
be less p27 remaining in the supernatant following Cdk4 immunodepletion in the lysate
from E2 treated cells than in lysate from ICI treated cells, which is consistent with this
proposal. To test whether there is an increase in Cdkl association with Cdk4 following
E2 treatment, the pellets from the sequential immunodepletions were analyzed by
Western blot (Figure 6B). In lysates of both ICI and E2 treated cells, equal amounts of
p21 co-immunoprecipitated with Cdk4, and equal amounts of Cdk4 and cyclin D1 co-
immunoprecipitated with p21, even though the overall level of p21 was decreased in the
E2 treated cells.

An increase in the amount of p27 co-immunoprecipitating with Cdk4 was
detected in lysates from E2 treated cells, although a high background was observed. We
investigated the binding of p27 to control antibodies and found that p27 binds non-
speciﬁcally to pre-immune goat IgG coated beads (Figure 6B, panel labeled “mock” ID,
p27 WB). No such non-speciﬁc binding was observed for cyclin D1, Cdk4 or p21 (not
shown). The increase in p27 association with Cdk4 in E2 treated cells was conﬁrmed in
a second independent experiment which is shown in the panel marked with an asterisk in
Figure 6B. Since the total amount of p27 in the lysate was not signiﬁcantly different in

lysates prepared from E2 and ICI treated cells, the reduction of p27 in lysates from E2

60

treated cells following immunodepletion of Cdk4 and the increase in p27 co-
immunoprecipitating with Cdk4 in E2 treated cells suggests that, in response to E2, p27 is

redistributed to Cdk4 containing complexes from Cdk2 or some other cellular pool.

Discussion

The experiments described herein were designed to investigate the mechanisms
by which E2 and ICI regulate Cdk4 activity in MCF-7 cells. As reported by others (2, 10,
31, 36), we observed transient increases in cyclin D1 protein levels between 6 and 18 h
after E2 treatment of ICI pre-arrested cells (Figure 2C). However, this increase in cyclin
D1 protein did not correlate directly with Cdk4 activity, which remained low until 24 h
after E2 treatment (Figure ZB). At 24 and 30 h after E2 treatment, when Cdk4 activity
was high, the levels of cyclin D1 in both the total cellular lysates and in complex with
Cdk4 were indistinguishable from those seen before E2 treatment, suggesting that under
these conditions, Cdk4 activity is not controlled solely by the total level of cyclin D1.

In contrast to cyclin D1 , p21 levels did correlate with Cdk4 activity in both ICI
and E2 treated cells (Figures 1 and 2), and the functional signiﬁcance of this correlation
was conﬁrmed by a combination of lysate mixing and immunodepletion experiments.
The initial mixing experiments shown in Figure 3 indicated that ICI treatment leads to
Cdk4 inactivation via the induction of an inhibitor, rather than by the loss of a positive
activator or by a post-translational modiﬁcation. The fact that immunodepletion of p21,
but not p27, removed the inhibitory activity from extracts of ICI treated cells (Figures 4
and 5) strongly implicates p21 as this inhibitor. Others have reported that p21 levels are

regulated by estrogen and antiestrogens, and shown that active Cdk2 complexes are free

61

of p21 in E2 treated MCF-7 cells (9, 36, 48). In this report, we conﬁrm that p21 levels
are regulated by ER signaling, and demonstrate for the ﬁrst time that p21 inhibits Cdk4
activity in antiestrogen treated MCF-7 cells.

Because p21 has been reported to play both activating and inhibitory roles in
regulating Cdk4 activity (5, 12, 21), and because we show that high levels of p21 protein
are associated with Cdk4 inactivation, it was of interest to investigate the composition of
the active Cdk4 complexes in E2 treated cells. The co—immunodepletion experiments
shown in Figure 6A demonstrate that the vast majority of the cyclin D1 is present in
complexes containing Cdk4 and p21 in both E2 and ICI- treated cells. This argues against
a model in which there is a large pool of active, “p21-free” cyclin D1/Cdk4 complexes in
E2 treated cells. However, it remains possible that the activity recovered is due to p21
free complexes which represent a small minority of the total Cdk4 complexes in the
lysate, and thus have evaded our detection. In fact, a similar situation has been reported
for Cdk2 in MCF-7 cells, in which active complexes that lack p21 can only be identiﬁed
after biochemical fractionation (3 6).

If both active and inactive Cdk4 complexes contain p21, there are several
alternative mechanisms by which it may regulate Cdk4 activity. Stoichiometry of
binding may determine whether p21 acts as a Cdk4 activator or inhibitor. A similar
model is proposed for p21’s regulation of cyclin A/Cdk2 complexes, although there is
disagreement as to its accuracy (1 , 12, 15, 26). In some of our experiments (see Figure
3C), less p21 appears to be bound to Cdk4 in E2 treated than in ICI treated cells, but this
has not been observed in every experiment. For example, in Figure 6B, the amount of

p21 co-precipitating with Cdk4 was indistinguishable in E2 and ICI treated cells. Since

62

p21 is a phosphoprotein (18), it is also possible that p21 may be differently modiﬁed in
E2 and ICI treated cells, and that such a modiﬁcation may modulate its Cdk4 inhibitory
activity. Finally, a p21-associated protein might be responsible for the regulation of
Cdk4 activity; a p21 associated factor has been reported which speciﬁcally modulates its
inhibition of cyclin E/Cdk2 activity (8). Future studies will seek to identify how estrogen
and antiestrogens regulate p21 levels, and how p21 regulates the activity of Cdk4 in
response to ER signaling. Because we have identiﬁed p21 as an important mediator of
antiestrogen’s effects in human breast cancer cells, we will also investigate whether a
defect in p21’s inhibition of Cdk4 could contribute to the development of antiestrogen
resistance.

Our results clearly demonstrate that the p21 which accumulates in ICI treated
cells inhibits Cdk4 activity. They also suggest that the decrease in p21 levels after E2
treatment is required for sustained Cdk4 activity. The data described above, together
with previous reports, suggest that an antiestrogen-induced cell cycle arrest is due to a
shift in the balance between activators (cyclins) and inhibitors (p21 or p27) of G1 Cdks.
Consistent with this idea, both overexpression of cyclin D1 (29, 35, 50) and ablation of
p21 or p27 expression (3) can cause G1 -)S progression in the presence of antiestrogen, at
least in the short term, in MCF-7 cells. In different model systems and in clinical cases
of breast cancer, different relative levels of activators and inhibitors may affect which
factor limits Cdk activity and ultimately proliferation. This in turn may inﬂuence the
responsiveness of ER positive breast tumors to antiestrogen therapies, and changes in the
levels or functions of Cdk activators and inhibitors during treatment may account for the

development of antiestrogen resistance.

63

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68

CHAPTER THREE
Induction of Cyclin D1 in MCF-7 Cells Supports Short Term

Proliferation in the Presence of Antiestrogen

69

Chapter 3: Induction of Cyclin D1 in MCF-7 Cells Supports Short Term

Proliferation in the Presence of Antiestrogen

ABSTRACT
Ampliﬁcation of the cyclin D1 gene and/or overexpression of its protein product is a
common feature of clinical breast cancer, and experimental evidence suggests that cyclin
D1 overexpression may allow estrogen receptor positive breast cancer cells to overcome
the G1 arrest imposed by antiestrogen treatment in vitro. To determine the mechanisms
by which cyclin D1 stimulates proliferation in the presence of antiestrogen and to
examine the potential contribution of cyclin D1 overexpression to antiestrogen resistance,
we describe a sub-clone of the MCF-7 human breast cancer cell line engineered to
conditionally express epitope tagged cyclin D1 upon the addition of a synthetic drug.
Using this novel gene regulatory system, we show that ectopic cyclin D1 expression can
be tightly regulated, and that cyclin D1 overexpression is sufﬁcient to allow cells arrested
in G1 phase by antiestrogen to enter S phase. Ectopic cyclin D1 can activate Cdk4 in the
presence of antiestrogen, but does not sustain long term Cdk4 activity or proliferation.
Ectopic cyclin D1 expression delayed, but did not prevent, antiestrogen mediated
induction of the Cdk inhibitor p21m‘mCipl and inhibition of Cdk4 activity in asynchronous
cells. These data suggest that cyclin D1 overexpression may contribute to antiestrogen
resistant growth of breast Cancer cells, but that additional alterations, such as bypassing

p21wamcipl, may be necessary for the acquisition of a stable antiestrogen resistant

phenotype.

70

Introduction

Cyclin D1, along with cyclins D2 and D3, are the regulatory partners of cyclin
dependent kinase 4 (Cdk4)2, a serine threonine kinase that phosphorylates the
retinoblastoma protein (Rb) (24, 28). Cyclin D1 association with Cdk4 facilitates Cdk4
nuclear localization and phosphorylation by the Cdk activating kinase (CAK) (11).
Hyperphosphorylated Rb contributes to cellular proliferation by relieving transcriptional
repression of E2F regulated genes (4, 46). The cyclin Dl/Cdk4 complex can also
associate with one or more cyclin dependent kinase inhibitor (Cdkl) such as p2 lwa'mCipl
(p21)(12, 19). Although p21 was originally identiﬁed as an inhibitor of cyclin D/Cdk4
activity(49) and it clearly plays this role in the context of ER signaling (41), recent work
has suggested that p21 may also facilitate cyclin D association with Cdk4 and enhance
catalytic activity (8, 50).

Recent investigations have suggested that cyclin D1 may be involved in the
development and progression of breast cancer. Cyclin D1 is encoded by the CCNDl
gene, located on chromosome 1 1q13, and this locus is ampliﬁed in approximately 15% of
breast tumors (13, 23, 37). Cyclin D1 protein overexpression is more common, occuring
in one third of breast cancers, suggesting that gene ampliﬁcation is not the only
mechanism by which cyclin D1 protein expression could be increased (15, 51). Gene

ampliﬁcation and protein overexpression both correlate positively with estrogen receptor

(ER) expression in breast cancer (13, 22, 42, 43, 51).

 

2Abbreviations used are: Cdk, cyclin dependent kinase. ER, estrogen receptor. p21, WAFl/Cipl. p27,
Kipl. p16, INK4a. Cdkl, Cdk inhibitor. Rb, retinoblastoma protein. E2, l7B-estradiol. AP, AP1510. IP,
immunoprecipitation. 26D, MCF-7-26D cell line. ICI, 1C1 182,780. DTT, dithiothreotol. IMEM,
improved modiﬁed Eagle’s medium. EGF-R, epidermal growth factor receptor. CAMP, cyclic adenosine
monophosphate. CSS, charcoal stripped serum. Minutes, m. Hours, h. FBS, fetal bovine serum.

71

The signiﬁcance of cyclin D1 in breast cancer is unclear. Positive
immunostaining and cyclin D1 gene ampliﬁcation are reported both to predict improved
relapse free survival (6, 36), and to have no association with tumor grade or prognosis
(42, 43). Elevated cyclin D1 mRN A and positive immunostaining correlated with a poor
prognosis if in combination with positive immunostaining for ERa, Rb or epidermal
growth factor receptor (EGF-R) (25, 29). These inconsistent results suggest that in
breast tumors, other factors may inﬂuence cyclin D1 ’3 effects on the course of disease, if
any.

Cell lines whose proliferation is dependent on estrogen have been utilized to
examine the relationship between cyclin D1 and ER signaling. Cyclin D1 mRN A and
protein are upregulated following E2 treatment of the breast adenocarcinoma derived
MCF-7 cell line, pre-arrested either by serum deprivation or antiestrogen treatment (1 ,
14, 27, 39), and E2 treatment causes cyclin D1 mRNA induction and Cdk4 activation in
E2 responsive tissue in vivo (2). Antiestrogen treatment is reported to reduce cyclin D1
mRN A and protein (44, 45), and this reduction precedes observable effects on Rb
phosphorylation and cell cycle, suggesting a causative role in inhibiting proliferation. E2
affects cyclin D1 transcription in MCF-7 and ZR-75 cells in a protein kinase-A
dependent manner through a CRE like sequence, and transcription is enhanced by
binding of c-Jun/ATF 2 heterodimers to these sequences. Cyclin D1 expression is also
controlled by ERa interaction with Spl transcription factors at Spl binding sites
upstream of the promoter (7, 35, 40). Interestingly, cyclin D1 has been shown to directly
interact with ERoc and enhance its transcriptional activity in the absence of ligand, and

this interaction is enhanced by cAMP (26, 32, 52, 53).

72

The link between estrogen and cyclin D1 in human breast cancer, and cyclin D1 ’8
known role as a mediator of proliferation, led some to hypothesize that overexpression of
cyclin D1 would confer estrogen independent growth in cell lines derived from breast
tissue. In the HBL-100 cell line, derived from normal mammary tissue, stable
transduction with cyclin D1 inhibited proliferation and tumorigenicity (18), while in the
breast tumor derived MCF-7 and T47D cell lines, conditional expression of ectopic
cyclin D1 caused cells arrested by serum deprivation or antiestrogen treatment to activate
cyclin E/Cdk2 complexes and enter the cell cycle (31, 38, 47). Although ectopic cyclin
D1 expression caused arrested cells to progress from G1 to S phase, it was not sufficient
to support long term growth in the presence of antiestrogens (34).

In the current work, we address the apparent paradox that cyclin D overexpression
causes G1 arrested breast cancer cells to progress to S phase in the presence of
antiestrogen but does not support long term proliferation in the presence of antiestrogen.
Utilizing a novel gene regulation system (3), Cunjie Zhang developed an MCF-7 derived
cell line that expresses an epitope tagged cyclin D1 protein upon addition of an otherwise
innocuous drug to the culture media. Consistent with previous reports, we show that
induction of cyclin D1 does cause cell cycle progression in antiestrogen arrested cells, but
is insufﬁcient to promote long term growth in the presence of antiestrogen. The ectopic
cyclin D1 is able to activate Cdk4 initially, however, Cdk4 activity is downregulated and
returns to basal levels within 72 h post treatment, despite the presence of abundant
ectopic cyclin D1 in stable association with Cdk4. We further demonstrate that the level
of the Cdkl p21, which is downregulated after antiestrogen arrested cells are treated with

estrogen, remains high in the presence of antiestrogen despite ectopic cyclin D1

73

expression. These data suggest that ER ligands exert control over the passage of MCF-7
cells through the G1 checkpoint by changing the balance between activators and
inhibitors of G1 Cdk complexes, and that cyclin D1 overexpression alone is an unlikely

cause of antiestrogen resistance in the absence of other changes.

Materials and Methods

Cell lines and culture conditions. MCF-7-26D cells were generated in two steps from
MCF-7 cells using Ariad Pharmaceuticals’ AP1510 gene regulation system (3) by Ms.
Cunjie Zhang. MCF-7-7-6 cells were generated by transfection of MCF-7 cells with
pCEN-F3p65/Z1F3; this is an expression vector encoding a G418 selectable marker, a
DNA binding domain fused to multiple FKBP (FK506 binding protein) domains, and a
transactivation domain fused to multiple FKBP repeats. MCF-7-7-6 cells were then
stably transfected with pLH-ZlZ-I-CCNDl—F LAG, which encodes hygromycin resistance
gene and a FLAG epitope tagged human cyclin D1 gene downstream of repeated unique
DNA sequence elements recognized by the DNA binding domain encoded by the ﬁrst
plasmid. After selection in hygromycin, one cell line, designated MCF-7-26D, exhibited
tight regulation of ectopic cyclin D1 expression in response to the dimerizing drug
AP1510 and was chosen for further charaterization. MCF-7-26D cells were cultured at
37° with 5% C02 in IMEM (BioFluids) with 5% fetal bovine serum (FBS, HyClone) and
penicillin/streptomycin (Gibco) and 10 pg/ml hygromycin (Gibco). Experiments were

performed in phenol red free IMEM (BioFluids) alone or containing 5% charcoal stripped

serum (CSS, HyClone), 10 nM 17B-estradiol (E2, Sigma), 10 nM ICI 182780 (ICI,

74

AstraZeneca), and/or 300 nM AP1510 (AP, Ariad Pharmaceutical) as indicated. For
experiments involving long term culture (Figure 4), media were changed at 72 h after

initial treatment.

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

was determined using ModF it and WinCycle LT software.

Immunoprecipitations and kinase assays. Immunoprecipitations (IPs) and Cdk activity
measurements were performed using modiﬁcations of a published method (28). 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, lmM NaVO4, 0.1 mM PMSF, 10 pg/ml leupeptin, and 2 pg/ml aprotinin). The
lysates were cleared by centrifugation, and protein concentrations were quantitated using
BioRad’s protein assay reagent. For IPs, 75 pg of total protein was diluted to 500 pl with
IP buffer, then incubated for 60 minutes (m) rocking at 4° C with 1.5 pg antibody (anti-

Cdk4 H-22-G, anti-Cdk2 M2, or normal goat IgG, all from Santa Cruz) bound to 7.5 pl

75

protein G agarose beads (Boehringer Mannheim). Beads were pelleted and washed four
times with 100 pl 1P 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 pg Histone H1 (Sigma) or 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 Rb were bound (GST-Rb bacterial expression vector was kindly provided by Dr.
William Kaelin of the Dana Farber Cancer Institute). 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
polyacrylamide gels, which were then dried and exposed to autoradiography ﬁlm. The
data were quantitated by phosphorimaging with a Storm phosphorimager (Molecular

Dynamics) using ImageQuant software.

Western blotting. Either 20 pg total cell lysates or aliquots of IP pellets were resolved

on 12% SDS-polyacrylamide gels, transferred to membranes and probed with antibodies

for cyclin D1 (UB1 06 137, Upstate Biotechnology), p21wan (p21-c-19, Santa Cruz
Biotechnology), cyclin A (14531C, Pharmingen) or actin (Sigma clone AC-40).
Membranes were then incubated with horseradish peroxidase conjugated goat anti-rabbit

(BioRad) or goat anti-mouse (American Qualex) secondary antibodies, and

76

immunoreactive proteins were detected using Super Signal West Pico Chemiluminescent

Substrate (Pierce).

Results

Generation of MCF - 7-26D cells. Cells which express ectopic, FLAG tagged cyclin D1
from a drug inducible promoter were generated using ARIAD Pharmaceutical’s AP1510
expression regulation kit (3) by Ms. Cunjie Zhang prior to the author’s arrival in the
Conrad lab. MCF-7-7-6 cells were generated by stable transfection of MCF-7 cells with
a tricistronic expression plasmid encoding a selection marker, a DNA binding domain
fused to multiple FKBP domains, and a transactivation domain fused to multiple FKBP
domains. Upon addition of the FK506 derivative AP1510 (AP), the two fusion proteins
dimerize and become a potent transcriptional activator at promoters containing unique
DNA sequences recognized by the DNA binding domain — FKBP ﬁision protein (Figure
1A).

MCF-7-26D cells were generated by stable transfection of MCF-7-7-6 cells with
an expression plasmid encoding a FLAG epitope tagged cyclin D1 under control of a
minimal IL-2 promoter downstream of repeated unique sequence motifs recognized by
the DNA binding domain encoded by the ﬁrst plasmid. A diagram of the strategy for
regulating ectopic cyclin D1 expression is shown in Figure 1A. Upon addition of AP to
the culture media, MCF-7-26D cells (26D cells) express ectopic cyclin D1 that is
recognized by antibodies to wild type human cyclin D, but distinguishable from

endogenous cyclin D due to its shiﬁed mobility in electorphoretic gels (Figure 18).

77

 

It
A- 'eguamy —CMVJ:I DNAB H TA H neo I——

plasmid: l l

 

target

plasmid: leZFHDl — IL-2 ——lﬂagl CCNDl l———

 

addition of

dimerizer:

 

 

 

 

8' FBS FBS+AP

c - 2: cyclin D1*

cyclin D

Figure 1: Construction of MCF - 7-26-D cells. A) Schematic of regulatory system used
to induce expression of ﬂag-tagged cyclin D1 in breast cancer cells. The regulatory
plasmid encodes two fusion proteins whose drug induced dimerization leads to
transcriptional activation of the target gene. CMV, cytomegalovirus promoter. DNAB,
DNA binding domain - FKBP fusion protein. TA, transactivation domain - FKBP
fusion protein. Neo, G418 resistance cassette. Hygro, hygromycin resistance cassette.
12xZFHD1, unique DNA sequence repeats recognized by DNAB. IL-2, minimal
interleukin-2 promoter. Flag, ﬂag epitope tag. CCNDl, human cyclin D1 cDNA. AP,
AP1510 dimerizer. RNAP, RNA polymerase. B) Regulated expression of ﬂag-tagged
cyclin D1 in MCF-7-26D cells. Cells were treated with either PBS or FBS plus the
dimerizer AP1510 for 24 h. Aliquots of lysates were Western blotted with a polyclonal
antibody which recognizes human cyclin D. The slower migrating band is ectopic, ﬂag
tagged cyclin D1.

78

Cell cycle eﬂects of cyclin D1 induction. To assess the effects of ectopic cyclin D1
expression on breast cancer cells’ ability to proliferate in the presence of antiestrogen, an
experiment was conducted in which 26D cells were pre-arrested with ICI in the presence
of charcoal stripped serum (CSS). The arrested cells were then washed and treated with
either E2 or ICI+AP1510, harvested at intervals over 30 h, and ﬁxed in ethanol. Cells
were then stained with propidium iodide and their DNA content evaluated by ﬂow
cytometry (Figure 2A). Prior to treatment (0 h), the cells were effectively arrested in the
G1 phase of the cell cycle. Estrogen treatment caused cells to re-enter the cell cycle by
24 h, and induction of cyclin D1 in the continued presence of ICI resulted in a 2.5 fold
greater increase in the percentage of cells in S phase. The fraction of cells entering S
phase after 24 h AP+ICI treatment varied among numerous experiments, but was
generally comparable to that achieved by E2 treatment without ICI. This suggests that
the amount of cyclin D1 protein may limit proliferation in the presence of antiestrogen.
We next asked whether ectopic cyclin D1 expression could override ICI’s effects
in the absence of other growth factors present in serum. Cells were plated in 5% fetal
bovine serum (FBS) for 24 h to allow attachment. The cells were then washed and
incubated in serum free media (SFM) for 48 h to induce quiescence, after which they
were washed and treated with SFM plus ICI, E2, AP and CSS, alone and in combination,
for 24 h. Cells were then harvested and their cell cycle phase distributions determined by
ﬂow cytometry as described above. As indicated in Figure 2B, serum deprivation caused
cell cycle arrest in 26D cells, with only 5% of cells in S phase. The addition of ICI did

not further depress the fraction of cells in S phase. Treatment with E2 alone induced

79

.>

I E2
[:1 ICI+AP

U1
0

b
O

N
0

percent of cells in S phase
(A)
C

d
O

 

0 h 6 h 24 h 30 h
time after treatment

percent of cells in S phase

 

SFM SFM SFM SFM SFM SFM C55 C55
+ICI +E2 +AP +AP +AP +E2
+ICI +E2

Figure 2: Effects of cyclin D1 expression on growth arrested cells. A) 26D cells
were pre-arrested with ICI for 48 h, then treated with either E2 or ICI+AP. Cells
were harvested at intervals over 30 h and analyzed for cell cycle distribution by ﬂow
cytometry. The average of two samples is shown; results are representative of three
repeated experiments. B) 26D cells were plated in 5% FBS, arrested by serum
deprivation for 48 h, then treated for 24 h with serum free media (SFM) alone or with
ICI, E2, or AP as indicated. Cells were also treated with 5% charcoal stripped serum
(CSS) alone and in combination with E2. Cells were collected and the percentage of
cells in S phase determined by ﬂow cytometry. The average of three samples is
shown; error bars indicate one standard deviation.

80

proliferation, with nearly 30% of cells in S phase. Induction of ectopic cyclin D1 via
treatment with AP caused a lesser degree of proliferation that was unaffected by the
addition of ICI, with approximately 17% of cells in S phase in both cases. Samples
treated with AP+E2 had a higher percentage of cells in S phase than samples treated with
either alone. The addition of CSS alone caused a slight increase in the percentage of cells
in S phase, and, as expected, the most robust proliferation was observed with the addition
of CSS and E2. These data suggest that in the absence of serum growth factors, cyclin
D1 levels limit proliferation, but that both E2 and serum have additional targets which

promote proliferation in breast cancer cells.

Activation of endogenous Cdk4 by ectopic cyclin D1. Inducing FLAG tagged cyclin D1
in 26D cells caused cell cycle progression in the presence of the antiestrogen ICI. We
hypothesized that this was due to the ectopic cyclin D1 binding to and activating Cdk4,
which then phosphorylated substrates critical for the initiation of DNA synthesis, such as
Rb. To test this hypothesis, 26D cells were pre-arrested in ICI for 48 h, washed, and
treated with either E2 or ICI + AP. Cells were harvested at intervals over 30 h, aﬁer
which whole cell lysates were prepared and analyzed using a Cdk4 in vitro kinase assay
in which Cdk4 is immunoprecipitated and incubated with recombinant Rb and
radiolabeled ATP.

E2 treatment caused a dramatic increase in Cdk4 activity that was evident by 24 h
after treatment (Figure 3A). Induction of cyclin D1 in the continued presence of ICI also
activated Cdk4 with somewhat faster kinetics than E2 treatment. To test whether ectopic

cyclin D1 could be detected in complex with Cdk4, lysates and reaction products from

81

c_L 2

. 0

hours after E2 L59» ICI+AP 9

treatment: 0 6 12 24 30 24 6 12 24 3O 24
GST-Rb —) .1... u I! I.

B.

 

 

50 :'__ I E2
40 __ I ICI+AP

 

 

 

 

 

 

 

N
O

 

fold induction
U0
0

  

_:
O

 

o 6 12 24 30
hours after treatment

C. lysates Cdk4 pellets
2

 

0 1224 12 24 012 24241224
cyclin D13 “M” m .9

actin -> ”was!“

0
E2 ICI+AP E2 2‘ ICI+AP

 

Figure 3: Ectopic cyclin D1 activates Cdk4 in the presence of antiestrogen. 26D
cells were pre-arrested with ICI for 48 h, then treated with either E2 or ICI+AP and
harvested at intervals over 30 h. Whole cell lysates were prepared and quantitated.
A) Aliquots of lysates were immunoprecipitated (IPd) with anti-Cdk4 or control (IgG)
antibodies. Pellets were reacted with radiolabeled ATP and recombinant Rb substrate
(GST-Rb). Products were resolved on gels and visualized by autoradiography. B)
Substrate bands from (A) were quantitated by storage phosphorimaging. Band
intensity is plotted relative to the untreated, 0 h sample. C) Aliquots of lysates (left
panels) and Cdk4 co-IPs (right panel) were Western blotted for human cyclin D1 and
actin. The slower migrating band in the cyclin D1 panel is ectopic, FLAG tagged
cyclin D1. The data shown in this experiment was repeated in a second experiment.

82

the kinase assay were Western blotted for cyclin D1 and actin. E2 treatment caused an
increase in cyclin D1 both in total and in complex with Cdk4 at 12 h after treatment, but
levels returned to pre-treatment levels by 24 h. Treatment with AP induced expression of
the slower migrating ectopic cyclin D1 at 12 h, and expression was sustained until 24 h.
The amount of ectopic cyclin D1 in complex with Cdk4 decreased from 12 h to 24 h, and
expression of ectopic cyclin D1 appeared to decrease the amount of endogenous cyclin
D1 both in total and in complex with Cdk4. Interestingly, although more cyclin D1 was
in complex with Cdk4 at 12 h in AP + ICI treated cells, activation was not observed until
24 h, when levels were lower. Although cyclin D1 may be limiting for Cdk4 activation,
increasing the amount of cyclin D1 in complex with Cdk4 does not cause immediate
activation of the complex, suggesting that further modiﬁcation of the Cdk4 complex must

occur prior to activation.

ICI treatment and ectopic cyclin D1 expression in asynchronous cells. The data
presented above demonstrate that ectopic cyclin D1 can activate Cdk4 in the presence of
ICI after a two day ICI pre-arrest. We next tested whether ectopic cyclin D1 could
sustain Cdk4 activity in proliferating cells treated simultaneously with ICI and AP. 26D
cells were plated in 5% FBS for one day, then treated with E2 for 48 h. Cells were then
washed and treated with either E2, ICI, or ICI + AP and harvested at intervals over 30 h.
Cellular lysates were prepared and analyzed by Cdk4 and Cdk2 kinase assay and
Western blotting.

The addition of fresh E2 to proliferating 26D cells caused a modest increase in

both Cdk4 and Cdk2 activities at 30 h (Figure 4). ICI treatment caused a decrease in

83

A' Cdk4 IP
hours after ICI ICI+AP E2_

treatments 0 6 18 30 6 18 30 30 303 _

 

196 IP

 

 

 

drum: W.» rm- IWWW m- .

foldchange 101.5 0602 14 1..305 1301

 

B. Cdk2 IP
% 2
hours after 9 g E2 :33

treatment: 0 30 3o 30 30
HistoneH13’ ‘

 

fold change: ~- 1 O OO 2 O. 5 1.9 O.2

C. ICI ICI+AP E2
9 618 39 618 .3930

cyclinD “mum‘s-4......

 

p21 «4...... _

2.12:3. J - ‘.-?~r-,-.\r.w'J‘mr‘-.-.-“i';rx""Hen-w. U. *1 .. ' --

Figure 4: Expression of ectopic cyclin D1 delays, but does not prevent, inhibition of
Cdk4 and Cdk2 activity by antiestrogen. 26D cells were treated with E2 for two days to
generate an asynchronous population. Cells were then treated with either ICI or ICI+AP
and harvested at intervals over 30 h. Whole cell lysates were prepared, quantitated and
analyzed for G1 Cdk activity and cyclin D, p21 and actin levels. A) Autoradiogram
showing Cdk4 in vitro kinase assay performed as described. Numbers below lanes
indicate the fold change relative to the 0 h activity as quantitated by storage phosphor
imaging. B) Autoradiogram showing histone H1 kinase activity of Cdk2 or control (IgG)
immunoprecipitates. Numbers below lanes indicate fold change relative to the 0 h
activity. C) Aliquots of lysates were Western blotted for cyclin D, p21, and actin.

84

Cdk4 activity by 18 h, and both Cdk4 and Cdk2 activities were at background levels by
30 h. Consistent with our earlier observations in MCF-7 cells (41), Cdk4 activity
decreased after ICI treatment without a corresponding decrease in cyclin D levels. Cells
treated with ICI + AP sustained high levels of Cdk4 activity through 18 h, but activity
was reduced to approximately half by 30 h, as was Cdk2 activity. The decrease in Cdk4
activity occurred despite continued high levels of ectopic cyclin D1. However, while
levels of the Cdkl p21 increased when cycling 26D cells were treated with ICI alone,
correlating with Cdk4 inactivation, there was a delayed and less dramatic increase in p21
when ICI was combined with cyclin D1 induction (Figure 4C). Cyclin D1 delayed, but
did not prevent, inhibition of GI Cdks in asynchronous cells treated with ICI, correlating

with delayed induction of p21.

Failure of ectopic cyclin D1 to support long term proliferation or Cdk4 activity in
antiestrogen. Others have reported that ectopic cyclin D1 expressed using a tetracycline
inducible system is insufﬁcient to promote long term proliferation in antiestrogen treated
breast cancer cells (34). To conﬁrm this ﬁnding, and to examine the mechanisms
responsible, we examined the effects of inducing cyclin D1 in the presence of ICI over
the course of several days treatment. 26D cells were pre-arrested in ICI as described,
then treated with either E2 or ICI+AP. Cells were collected at 24 h intervals over four
days and analyzed for cell cycle distribution by ﬂow cytometry as described. As
indicated in Figure 5A, cells were efﬁciently arrested prior to treatment, with 78% of
cells in G1 phase. Consistent with our earlier results (Figure 2A), both E2 and ICI+AP

treated cells entered the cell cycle by 24 h after treatment, with E2 treated cells reduced

85

.>

§ ; m —~
5 80 a 301 E2
o. 1 .1: 1
G 70 1 5} j I ICI+AP
: 60 1 E Z
'=: 50 £203 '
8 40 3”: . 1
:6 30 8 1
5810 ,, g 1
0 24 72 96 0 24 48 72 96
hours after treatment hours after treatment
B E E
o 0 K9
E2 9 ICI+AP 9 lCl

hours after ——
treatment: f 0 24 72 96 120' 24

    

:1. 72 96.120. 24. ‘29...

      

 

GST—Rb —)

C.

lysates Cd k4 Co-IP
hours after ,E2

,2, ,, ,lCliAP ICI 132 ilCl+AP ,, ICI
treatment: 0 24 2296120 23.21296 120 120 0 24 72 96 120 24 72 96 120 120

 

c.

 

actin mu“ ,

Figure 5: Expression of exogenous cyclin D1 does not support long term proliferation or
Cdk4 activation in the presence of antiestrogen. A) 26D cells were pre-arrested in ICI,
then treated with either E2 or ICI+AP and harvested at 24 h intervals for 96 h. Cell cycle
phase distribution was determined by ﬂow cytometry as described. The average of three
samples is shown; error bars indicate standard deviation. B) 26D cells were pre-arrested
in ICI, treated with either E2, ICI+AP, or ICI and harvested at the indicated times aﬁer
treatment. Lysates were prepared, quantitated, and Cdk4 in vitro kinase activity was
determined as described. C) Aliquots of lysates and Cdk4 IP pellets from (B) were
Western blotted for cyclin D, p21 and actin.

86

to 46% G1 phase and ICI+AP treated cells to 60% G1 phase; both treatments showed a
corresponding increase in the percentage of cells in S phase (not shown). The fraction of
G1 phase cells in both treatments increased after 24 h. While the percentage of G1 phase
cells leveled off at approximately 60% in E2 treated cells, consistent with an
asynchronous population, ICI+AP treated cells appeared to re-arrest in G1 phase by 96 h
after treatment, with 80% of cells in G1 phase. These data are consistent with others’
observations that induction of ectopic cyclin D1 in the presence of antiestrogen can cause
progression from G1 to S phase, but is insufﬁcient to sustain long term cell growth (34,
38, 47, C. Zhang, unpublished observations).

Induction of cyclin D1 did not prevent, but appeared to delay, the inhibition of
Cdk4 activity and the increase in p21 levels seen after ICI treatment of proliferating cells
(Figure 4). Previously, we showed that p21 acts as an ICI regulated inhibitor of Cdk4 in
MCF-7 cells (41). We therefore hypothesized that the failure of ectopic cyclin D1 to
support long term proliferation was due to the inability of ectopic cyclin D1 — Cdk4
complexes to overcome inhibition by p21 after an initial activation. To test whether
Cdk4 was inactivated after long term induction of ectopic cyclin D1 , 26D cells were pre-
arrested in ICI and then treated with either E2 or ICI+AP. Cells were harvested at
intervals over ﬁve days and analyzed by Cdk4 kinase assay and Western blotting (Figure
5B, C). E2 treatment caused activation of Cdk4 by 24 h after treatment, and activity was
sustained for 120 h; this activation was not accompanied by any changes in the level of
total cyclin D1 protein, although there was a transient increase in Cdk4 associated cyclin
D1 at 24 h post treatment. Levels of p21 in the lysates decreased by 24 h after E2

treatment and continued to decrease through 120 h.

87

Induction of ectopic cyclin D1 in the presence of ICI caused activation of Cdk4 to
about half the level of E2 treated cells by 24 h after treatment in this experiment. Unlike
E2 treated cells, in which Cdk4 activity was sustained, in ICI+AP treated cells Cdk4
activity began to decrease by 72 h after treatment and was undetectable by 96 h, despite
the presence of abundant ectopic cyclin D1 in the lysate and in complex with Cdk4.
Levels of p21 decreased to approximately the same level as with E2 treatment by 24 h
after treatment, but then increased gradually over the 120 h, coinciding with Cdk4
inactivation. These data suggest that the failure of ectopic cyclin D1 to support long term
cell growth in ICI is due to its inability to sustain activation of Cdk4, and that this is due
to increased and/or maintained levels of p2 1. This phenomena was observed in two
independent experiments.

Interestingly, when cells were treated with ICI alone for 120 h (in addition to the
48 h pre-arrest), endogenous cyclin D1 and p21 were not detectable in the lysate, and
cyclin D was barely detectable in complex with Cdk4; no Cdk4 activity was detected in
this sample (Figure 5B, rightmost lane). This suggests that p21 and cyclin D1 are
coordinately regulated in long term ICI treated cells, or, alternatively, that cell death

pathways have been activated which cause non-speciﬁc protein degradation.
Discussion
Breast cancer is an important disease whose etiology is associated with exposure

to estrogen and with increased expression of cyclin D1. The work described above

describes the consequences of expressing FLAG epitope tagged cyclin D1 in estrogen

88

dependent human breast cancer cells. We demonstrate that inducing ectopic cyclin D1 in
G1 arrested cells causes progression to S phase in the presence of antiestrogen, in both
the presence and absence of serum growth factors, although cells re-arrest in G1 aﬁer
several days of treatment. Cdk4 is activated following AP treatment in the presence of
ICI, and ectopic cyclin D1 co-precipitates with active Cdk4 by 24 h after induction.
However, Cdk4 is inactivated after long term AP+ICI treatment, despite the presence of
both ectopic and endogenous cyclin D1 in complex with Cdk4. The inactivation of Cdk4
coincides with a maintenance and/or gradual accumulation of p21 in the cells. We
propose that initially, ectopic cyclin D1 can bind Cdk4, forming p21 free cyclin D1-Cdk4
complexes that are active Rb kinases. At later time points, treatment with antiestrogen,
p21 levels increase until all cyclin D1-Cdk4 complexes contain and are inactivated by the
Cdkl. In the absence of Cdk4 activity, progression through the G1 checkpoint is
inhibited, and cells re-arrest.

This work provides an explanation for the somewhat disparate results of two
groups who have published reports characterizing cyclin D1 induction in stably
transfected MCF-7 derivatives. Sutherland et a1, using ectopic cyclin D1 expressed from
a zinc inducible promoter, showed that inducing cyclin D1 in the presence of antiestrogen
caused activation of Cdk4 and progression through at least one cell cycle, with concurrent
activation of cyclin E-Cdk2 complexes (3 8, 47). While Weisz et al also observed
progression through the G1 checkpoint when ectopic cyclin D1 expression was induced
from a tetracycline inducible promoter, they showed that antiestrogen inhibited long term
cell growth despite high levels of cyclin D1 (34). Our data corroborate the work of both

groups in an independent system of cyclin D1 induction in human breast cancer cells. In

89

addition, they provide a mechanistic explanation for these results: the failure of p21 to be
downregulated in the absence of E2.

The regulation of p21 is complex, and numerous signaling pathways converge at
the p21 promoter. Growth arrest by interferon-y and differentiation by interleukin-6 is
mediated by STAT activation of the p21 promoter (5, 9, l7), and epidermal growth factor
(EGF) mediated STAT activation of p21 expression is also reported in cells whose
growth is inhibited by EGF (33, 48). Transforming growth factor-B increases p21 levels
by acting through Spl and Sp3 transcription factors, while Myc represses p21 levels,
possibly by interfering with Spl and Sp3 activity (10, 16, 30). Transcription from the
p21 promoter is also controlled by E2F1 (21). Coordinate regulation of p21 and cyclin
D1 has been reported (20), and E2 can regulate transcription at the cyclin D1 promoter
through Spl sites (7), a potential point at which their regulation may intersect.

We suggest that p21 expression is increased or activated by ICI in estrogen
dependent breast cancer cells, and that in the presence of ICI sufﬁcient p21 is present to
inhibit the activity of all cyclin D1-Cdk4 complexes. This inhibition may be overcome
temporarily by expression of ectopic cyclin D1, but over time p21 levels are adjusted so
that all cyclin D1-Cdk4 is inactivated. In this model, the activity of Cdk4 and ultimately
cell proliferation is controlled by a balance of activating factors (D type cyclins) and
inhibitors (e. g. p21). In our system of antiestrogen mediated growth arrest it appears that
the cell has some mechanism to re-adjust the balance when it is altered experimentally.
This suggests that in human breast cancer, overexpression of cyclin D1 is unlikely to
result in the development of antiestrogen resistance in the absence of other changes. The

observation that cyclin D1 overexpression is a common feature of breast cancers and that

90

its overexpression often correlates with positive ER status may reﬂect cyclin D l ’s
involvement in other phases of tumorigenesis besides the development of antiestrogen

resistance (15, 51).

91

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CHAPTER FOUR

Characterization of LCC9, an Antiestrogen Resistant Derivative of MCF-7 Cells

97

Chapter 4: Characterization of LCC9, an Antiestrogen Resistant Derivative of

MCF-7 Cells

ABSTRACT
Although antiestrogen therapies represent a key advance in the treatment of breast cancer,
acquired resistance to antiestrogens is a major cause of treatment failure. The molecular
changes underlying the conversion from an antiestrogen sensitive to an antiestrogen
resistant phenotype are poorly understood, despite much investigation. The LCC9 cell
line, which is resistant to growth arrest by the pure steroidal antiestrogen ICI 182780, is a
derivative of the estrogen receptor positive, antiestrogen sensitive human breast cancer
cell line MCF-7. Here we compare the molecular events at the G1 -)S phase checkpoint
in LCC9 cells with their parental MCF-7 cells. Using transfection with estrogen receptor
response element driven luciferase expression vectors, we demonstrate that LCC9’s
estrogen receptor is regulated similarly to MCF-7’s. While MCF-7’s G1 phase cyclin
dependent kinases Cdk2 and Cdk4 are inactivated following antiestrogen treatment, we
show that both kinases remain active in antiestrogen treated LCC9 cells. The Cdk4
complexes from LCC9 cells retain sensitivity to inhibition by lysate from antiestrogen
treated MCF-7 cells. We show that compared to MCF-7, LCC9 cells have elevated levels
of cyclin D1 protein. In addition, while levels of the Cdk inhibitor p21wamCipl increase
after two days of antiestrogen treatment in MCF-7 cells, p21 is not highly regulated by
antiestrogen in LCC9 cells. These experiments suggest that in LCC9 cells, altered
signaling pathways upstream of cyclin D and p21 may maintain the activities of G1 Cdks,

allowing proliferation in the presence of antiestrogen.

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Introduction

Despite advances in detection and treatment, breast cancer remains a common and
deadly malignancy in the United States. Because many breast tumor cells require
estrogen for growth, drugs that block the effects of estrogen have long been used as
therapies for breast cancer patients. Tamoxifen, which acts as an antagonist of the
estrogen receptor (ER)3 in breast tissue, has been a successful treatment for ER positive
breast cancer and has recently been recommended for the prevention of breast cancer in
women at high risk for developing the disease. However, the usefulness of Tamoxifen
and other antiestrogens is limited by the development of resistant tumors in patients who
initially responded positively to therapy (33, 34, 72). The process by which ER positive
tumors acquire an antiestrogen resistant phenotype is unknown, and there are no
molecular markers associated with its development, with the possible exception of Her2
overexpression (50). Research in this area may point to diagnostic markers that predict
the effectiveness of antiestrogen therapies, or to alternative treatment strategies.

In sensitive tissues, the most biologically potent form of estrogen, 17B-estradiol
(E2), acts as a mitogen by binding to the ER, a nuclear steroid receptor. There are two
forms of ER, the classical ERa and the more recently described ERB (42, 65). Both ER
types act as a ligand activated transcription factors by direct binding to DNA at estrogen

response elements (EREs) and variations thereof and recruiting transcriptional

 

3 Abbreviations used are: ER, estrogen receptor. ERE, estrogen receptor response element. p21,

WAF l/Cipl. p27, Kipl. Cdk, cyclin dependent kinase. Cdkl, Cdk inhibitor. pr, retinoblastoma protein.
E2, 17B-estradiol. IP, immunoprecipitation. ICI, ICI 182,780. DTT, dithiothreotol. IMEM, improved
modiﬁed Eagle’s medium. CSS, charcoal stripped serum. Luc, luciferase. TK, thymidine kinase minimal
promoter. RT-PCR, reverse transcriptase-polymerase chain reaction. Minutes, m. Hours, h. FBS, fetal
bovine serum. EGF, epidermal growth factor. STAT, signal transducers and activators of transcription.

99

coactivator proteins such as CBP/p300, p/CAF, p160, and SRCl to the promoter region
(9, 44, 48, 64, 70, 71). Additionally, ER can act to modulate transcription in the absence
of DNA binding via protein — protein interactions with transcriptional activators such as
Apl and Spl (30, 56); in both cases, ER recruits to coactivators to the promoter, thereby

enhancing transcription. ERa and B have different activities at AP] sites (45), and

evidence suggests that ERB may act to inhibit transcription of some ERa regulated genes
(40). The activities of both ERs can be further regulated by phosphorylation by Erk, a
MAPK (1, 36, 66). Thus, evolution has produced a complex signaling network through
which estrogen mediated gene expression can be ﬁnely tuned.

In general, antiestrogens mimic the three dimensional conformation of estrogen
and bind ER’s ligand binding domain but do not allow transcriptional activation;
however, some antiestrogens have tissue and/or promoter speciﬁc agonist activity. For
example, Tamoxifen acts as an ER antagonist in the breast but has partial agonist activity
in the uterus, and has opposite activities at AP-l dependent promoters with the two
known ER isoforrns, ERa and ERB (20, 26, 45, 67). The “pure” antiestrogen ICI
182,780 (ICI), used in the present study, acts solely as an ER antagonist and can be an
effective second line antiestrogen therapy in patients whose tumors have become
Tamoxifen resistant (15, 16, 25 , 47). Resistance to pure ER antagonists occurs clinically
and in experimental models, although the effectiveness of pure antagonists in Tamoxifen
resistant breast cancer suggests that distinct mechanisms are responsible for resistance to
partial agonists and pure antagonists.

Several mechanisms have been proposed to explain the acquisition of antiestrogen

resistance. These include a loss of ER or selection of ER negative cells from a

100

heterogeneous tumor population, a change in ER function, altered metabolism of
antiestrogen, and inappropriate activation of estrogen dependent or independent growth
stimulatory signaling pathways. These alterations could occur alone or in combination
and could result from genetic lesions or epigenetic changes (13, 14, 23, 31, 46, 54).
While ER loss may be a cause of antiestrogen resistance prior to treatment, acquired
resistance during the course of treatment occurs in most cases without loss or mutation of
ER (32), and antiestrogen metabolism has not been shown to be a likely cause of
antiestrogen resistance (43). The composition of co—activators and/or co-repressors
interacting with antiestrogen bound ER may inﬂuence whether the ligand behaves as a
transcriptional activator or repressor at any given promoter, and changes in the makeup of
ER containing complexes may underlie the acquisition of antiestrogen resistance (19, 60,
63), although clinical evidence has been lacking thus far (10). Much recent attention has
therefore been placed on alterations in signal transduction pathways regulating cellular
proliferation as potential causes of antiestrogen resistance.

Estrogens and antiestrogens affect proliferation in target cells at the G1
checkpoint (18, 62, 68), passage through which is governed by the Rb pathway (see
Chapter 1, Figure 2). In cells containing wild type retinoblastoma protein (Rb),
progression through Gl to S phase is controlled by the activity of two types of
serine/threonine kinases, cyclin dependent kinase-2 (Cdk2) and Cdk4/6, which integrate
the activities of multiple signaling pathways. Cdk2 can form a catalytically active
enzyme when bound in one to one stoichiometry with either cyclin E or A, while Cdk4
and the related Cdk6 are activated by association with a D-type cyclin (27, 28, 57). In

addition to cyclins, Cdks can stably associate with Cdk inhibitors (Cdkls), including

101

1921“”cipl (p21) and p27k‘P' (p27). Originally described as inhibitors of Cdk activity,
recent data suggests that under some conditions some Cdkls can act to increase the
activity of cyclin D/Cdk4 complexes (11, 22, 52, 58, 73). Estrogen has been shown to
increase cyclin D1 protein levels, Cdk4 and Cdk2 activity, and Rb phosphorylation, and
to decrease Cdkl expression and association with Cdk2 in breast cancer cells (1 7, 18, 51,
53), and antiestrogen reverses these effects (7, 59, 68, 69). Inappropriate activation of G1
Cdks could therefore contribute to antiestrogen resistant grth of breast cancer cells.

In this work, we examine the molecular events underlying the G1-)S phase
transition in a model of acquired resistance to the pure steroidal antiestrogen ICI. The
MCF-7 cell line, derived from a human breast adenocarcinoma, is ERa positive, estrogen
dependent and antiestrogen sensitive (4, 5, 24, 61). Through in viva selection for
estrogen independent tumorigenicity and in vitro selection for grth in the presence of
increasing concentrations of ICI, Clarke et al generated the LCC9 cell line. LCC9 cells
retain ER, but are able to proliferate in concentrations of ICI that completely inhibit
MCF-7 cell growth (6). Below we compare signaling events in ICI and E2 treated MCF-
7 and LCC9 cells, demonstrate that LCC9 cells have numerous changes in the Rb
pathway, and that the causes of these changes are likely to be upstream of G1 Cdk

activation.

Materials and Methods

Cell lines and culture media. MCF-7 and LCC9 cells were obtained from Dr. Michael

Johnson of the Lombardi Cancer Center, Georgetown University. They were routinely

102

passaged in IMEM (BioFluids), supplemented with 5% fetal bovine serum (F BS,
HyClone), 100 units/ml penicillin and 100 ug/ml streptomycin (Gibco). In the
experiments described in Figures 1 and 2, early passage LCC9 cells were split into two
populations: one was cultured routinely in 5% F BS as described above, while the other
was routinely cultured in IMEM without phenol red (BioFluids) containing 5% charcoal
stripped serum (CSS, HyClone), penicillin / streptomycin and 10'8 M ICI (Astra Zeneca).
These two populations were cultured in this way for 7 passages (28 days) prior to the
experiments; the two populations were passaged at the same ratio and intervals. For
estrogen and antiestrogen treatments, cells were cultured in IMEM without phenol red
(BioFluids) containing 5% charcoal stripped serum (CSS, HyClone) and penicillin /
streptomycin, with either 10'8 M E2 (Sigma) or 10'8 M ICI (Astra Zeneca) unless
otherwise indicated. Cells were washed twice with PBS before all media changes. Cells

were cultured at 37° C with 5% C02.

Cell cycle analysis. MCF-7 and LCC9 cells were plated at 2.5 x 105 cells per 60 mm
plate in 5% F BS. After the indicated treatments, cells were trypsinized, washed in
phosphate buffered saline (PBS), suspended in PBS+10% F BS, ﬁxed with 80% cold
ethanol and stored at —20° 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-IOO. Cells were then analyzed for red ﬂuorescence on a FACSVantage
ﬂow cytometer; cell cycle distribution was determined using ModFit software. Three 60

mm plates were analyzed for each time point.

103

Transfections and luciferase assays. This experiment was performed by Dr. Hemant
Varma. MCF-7 and LCC9 cells were plated at 5x105 cells/60mm plate and transfected
with plasmid DNA using the Lipofectin reagent following the protocol provided by the
manufacturer (GIBCO-BRL). 1 ug of ERE-tk-luc or tk-luc (vector) plasmid was
cotransfected with 0.1 ug of pCMV-B-galactosidase as a control for transfection
efﬁciency (luciferase plasmid provided by Dr. Richard Miksicek; B-gal plasmid provided
by Dr. Richard Schwartz). Cells were kept in 5% charcoal dextran stripped serum (CSS)
containing medium for 24 hours followed by CSS alone or with ICI (lOOnM), E2 (lnM)
or E2 +ICI for an additional 24 hours. Cells were harvested at 48 h post-transfection and
both the luciferase and B-galactosidase activities were measured using the detection
reagents and protocols provided by the manufacturer (Clontech) on a Turner TD 20E
luminometer (Turner Designs). For each experiment, ERE-luciferase and B-galactosidase

transfections were performed in triplicate; tk-luc transfection was performed twice.

Immunoprecipitations and kinase assays. Immunoprecipitations (IP) and Cdk activity
measurements were performed using a modiﬁcation of a published method (41). 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, lmM NaVO4, 0.1 mM PMSF, 10 ug/ml leupeptin, and 2 ug/ml aprotinin). The

lysates were cleared by centrifugation, and protein concentrations were quantitated using

104

BioRad’s protein assay reagent. For IPs, 75 ug of total protein was diluted to 500 1.11 with
IP buffer, then incubated for 60 minutes (m) rocking at 4° C with 1.5 ug antibody (anti-
Cdk4 H-22-G, anti-Cdk2 M2 or normal goat IgG, Santa Cruz) bound to 7.5 ul protein G
agarose beads (Boehringer Mannheim). Beads were pelleted and washed four times with
100 pl IP buffer and twice with 100 ul 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 ul kinase buffer containing 10 uCi y-
[32P]ATP and 20 uM cold ATP. For Cdk4 assays, 1.0 ul glutathione sepharose 48

(Amersham Pharmacia) to which approximately 2.0 ug of a fusion protein between
glutathione-S-transferase (GST) and amino acids 792 to 928 of human Rb were bound
(GST-Rb bacterial expression vector was kindly provided by Dr. William Kaelin of the
Dana Farber Cancer Institute) was added as substrate. For Cdk2 assays, 2.0 ug histone
H1 (Sigma) was used as substrate. 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 polyacrylamide gels, which
were then dried and exposed to autoradiography ﬁlm. The data were quantitated by
phosphorimaging with a Storm phosphorimager (Molecular Dynamics) using

ImageQuant software.

Western blotting. Either total cell lysates (20 ug), IP supematants or IP pellets were

resolved on 12% SDS-polyacrylamide gels, transferred to membranes and probed with

antibodies for cyclin D1 (UB1 06 137, Upstate Biotechnology), cyclin E (14761C,

105

Pharmingen), p21wan (p21-c-19, Santa Cruz Biotechnology), p27"ipl (C-19, Santa Cruz
Biotechnology), Cdk4 (H22, Santa Cruz Biotechnology), Cdk2 (M2, Santa Cruz
Biotechnology), or actin (Sigma clone AC-40). Membranes were then incubated with
horseradish peroxidase conjugated goat anti-rabbit (BioRad) or goat anti-mouse
(American Qualex) secondary antibodies, and immunoreactive proteins were detected
using Super Signal West Pico Chemiluminescent Substrate (Pierce) according to

manufacturers’ protocol.

Lysate mixing assays. MCF-7 and LCC9 cells were treated as indicated, then harvested,
stored, and lysed as described above. Seventy ﬁve pg of protein from E2 treated MCF-7
or LCC9 cells were mixed with 7.5 to 75 ug of protein from ICI treated MCF—7 cells, and
diluted to 500 ul with IP buffer. The mixtures were incubated at 30° C for 30 m with
occasional agitation. Unmixed control reactions were diluted and incubated
simultaneously. The samples were then subjected to the Cdk4 kinase assay as described
above. Phosphorylated substrate band intensities were quantitated by storage

phosphorimaging.

Results

LC C 9 cells are not arrested by the antiestrogen 1C] 182 780. To verify the reported
antiestrogen resistant phenotype of LCC9 cells (6), we compared the cell cycle proﬁle of

MCF-7 and LCC9 cells treated with the antiestrogen ICI over a 42 h time course. To

evaluate the stability of this phenotype, two parallel populations of LCC9 cells were

106

 

 

 

 

% cells in 61 phase

 

 

18
hours after ICI treatment

 

 

 

% cells in S phase

 

 

 

 

 

 

O 6 18 24 30 42

hours after ICI treatment
Figure 1: The LCC9 cell line's antiestrogen resistant phenotype is stable in the
absence of selective pressure. MCF-7 cells (black bars) and LCC9 cells cultured long
term (> 1 month) in the presence of FBS (grey bars) or CSS+100 nM ICI (white bars)
were cultured in FBS for two days. Cells were then treated with 100 nM ICI and
harvested at intervals over 42 h. Cells were ﬁxed in ethanol, stained with propidium
iodide, and analyzed by ﬂow cytometry. A) The average percentage of cells in G1
phase; error bars indicate on standard deviation (n = 3). B) The percent of cells in S
phase.

107

used, one cultured long term in 10 nM ICI and the other cultured long term in 5% F BS.
Cells were plated at low density in FBS, then washed twice with PBS and treated with 10
nM ICI. Cells were harvested and ﬁxed at the times indicated, then DNA content was
measured with propidium iodide staining and ﬂow cytometry.

Both MCF-7 and LCC9 cells were asynchrous at the beginning of the time course,
with 38% of MCF-7 cells and approximately 50% of LCC9 cells containing a G1 phase
DNA content (Figure 1). MCF-7 cells began to accumulate in G1 phase after ICI
treatment, reaching nearly 90% G1 in phase by 42 h, with a corresponding reduction in
the fraction of cells in S phase. In contrast, LCC9 cells were unaffected by ICI treatment.
The fraction of cells in G1 phase was about 50% at each time point for both LCC9
populations. These data conﬁrm that LCC9 cells are resistant to the cytostatic effects of
the pure ER antagonist ICI, and demonstrate that LCC9’s antiestrogen resistant

phenotype is stable in the absence of selective pressure.

LC C9 's ER is regulated normally at a consensus ERE. Because LCC9 cells contain EROt
but have altered expression of E2 regulated genes (6), we speculated that LCC9’s ERor
may have an altered response to antiestrogen, and this might account for its antiestrogen
resistant phenotype. This possibility was tested by comparing MCF-7 and LCC9 in a
luciferase assay measuring transcriptional activity from a consensus ERE. Cells were
transfected with either ERE (ERE-tk-luc) or minimal promoter (tk-luc) plasmids and a B-
galactosidase control plasmid, then treated with CSS alone or containing either 100 nM

ICI, 1 nM E2, or 100 nM ICI + 1 nM E2.

108

 

-- ClERE-luc
5 . lTK-luc

 

 

 

1“
J
1
I—-}—-1

 

luc/b-gal

N
J

—l
i

 

 

 

 

 

css ICI £2 E2+|C| css ICI £2 E2+ICI
MCF-7 LCC9

 

 

Figure 2: LCC9 cells’ estrogen receptor is regulated normally by E2 and ICI.
MCF-7 and LCC9 cells were transfected with vectors encoding luciferase under the
control of either a consensus estrogen response element upstream of a minimal
promoter (ERE-luc) or the minimal promoter alone (TK-luc). Cells were co-
transfected with a B-galactosidase expression vector. Transfected cells were cultured
in charcoal/dextran stripped serum (CSS) for 24 h, then treated with either fresh
CSS, 100 nM ICI, 1 nM E2, or 100 nM + 1 nM E2 for an additional 24 h. Cells
were harvested and luciferase and B-galactosidase activities determined on a Turner
luminometer. The graph shows luciferase activity divided by B-galactosidase
activity. The experiment was performed three times, and three samples were
measured for each experiment. Error bars indicate one standard deviation (n = 9).

109

The luciferase activity divided by B-galactosidase activity to correct for possible
differences in transfection efﬁciency is shown in Figure 2. In MCF-7 cells, E2 treatment
led to strong transcriptional activation of the ERE, but not the control promoter. This
activation was abolished by co-treatment with ICI, conﬁrming its function as an E2
antagonist. Identical results were obtained in LCC9 cells, suggesting that LCC9’s ability
to proliferate in ICI is not due to a failure of ICI to downregulate transcription at
consensus EREs. Interestingly, the basal levels of ERE luciferase expression were

slightly but reproducibly higher in LCC9 than in MCF-7 cells.

C dk2 and Cdk4 are not inactivated by antiestrogen in LCC 9 cells. We and others have
shown that in MCF-7 cells, G1 Cdks are inactivated following ICI treatment (7, 59, 68).
To test whether LCC9’s ability to proliferate in the presence of ICI could be due to a lack
of Cdk inactivation, we compared the Cdk2 and Cdk4 associated kinase activities in ICI
treated MCF-7 and the two populations of LCC9 cells described in Figure 1. Cells were
plated in FBS for two days, then treated with ICI and harvested at the time points
indicated. Cellular lysates were immunoprecipitated with antibodies reactive to Cdk2 or
Cdk4; pellets were washed extensively and reacted with radiolabeled ATP and substrate
(histone H1 for Cdk2 and GST-Rb for Cdk4). Reaction products were resolved
electrophoretically and visualized by autoradiography.

As indicated in Figure 3A, Cdk2 in vitro kinase activity was inhibited by ICI
treatment by 18 h in MCF-7 cells, and was undetectable by 30 h. ICI inactivated Cdk4
activity in MCF-7 cells with similar kinetics (Figure 33). However, consistent with their

growth phenotype observed in Figure 1, neither G1 Cdk activity was downregulated in

110

A- ‘5» MCF-7 LCC-9(FBS) LCC-9(ICI)
00 6182430 0 6182430 0 6182430

 

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B. “.3. MCF-7 LCC-9(FBS) LCC-9(ICI)
0 0 61824300 618 24300 6182430
”dr- ﬁﬂﬁﬁﬁmnggw GST-Rb
C.

MCF—7 LCC-9(FBS) LCC-9(ICI)
01830 01830 01830

 

.- a- . - up a. - g .- p21(Cdk4co-IP)

w “we?” “M” w .1” «but W diam"?! 'rt‘r‘lﬂﬂi‘

actin

Figure 3: LCC9's GI Cdks are not inactivated by antiestrogen. MCF-7 and LCC9
cells (cultured for one month in either FBS or ICI as in Figure 1) were incubated in
FBS for two days, then treated with 100 nM ICI. Cells were harvested at the indicated
intervals and lysates were prepared and normalized. A) Aliquots of lysates were
immunoprecipitated with anti-Cdk2 antibodies. The pellets were incubated with
radiolabeled ATP and histone H1 substrate, and kinase reaction products were resolved
on SDS-PAGE and visualized by autoradiography. B) Aliquots of lysates were
immunoprecipitated with anti-Cdk4 antibodies, and the pellets were incubated with
radiolabeled ATP and recombinant retinoblastoma protein (GST-Rb) substrate.
Products were resolved on SDS-PAGE and visualized by autoradiography. C)
Aliquots of lysate and reaction products from (B) were analyzed by Western blot using
cyclin D, p21, and actin antibodies.

111

LCC9 cells following ICI treatment during the course of this experiment. While the
levels of Cdk2 activity were comparable in asynchronous MCF-7 and LCC9 cells (0 h),
LCC9 cells had reduced Cdk4 activity compared to MCF-7 cells. There were no
differences between the FBS-cultured and ICI-cultured LCC9 populations’ response to
ICI treatment. These results suggest that the defect in ICI signaling in LCC9 cells is
upstream of both G1 phase Cdks.

We next compared the levels of cyclin D1 and p21 protein in whole cell lysates
and in Cdk4 immunoprecipitates by Western blotting (Figure 3C). In MCF-7 cells, total
and Cdk4 associated cyclin D1 protein decreased slightly following ICI treatment, while
p21 levels were unchanged, although p21 is upregulated upon longer treatment in MCF-7
cells (see Figure 5). In LCC9 cells, levels of cyclin D were unchanged over the course of
30 h, and p21 appeared to decrease slightly. However, the amount of both cyclin D1 and
p21, in lysates and in Cdk4 pellets, was slightly but consistently higher in LCC9 cells

than in MCF-7 over numerous experiments.

Cdk4 activity in LCC9 is sensitive to inhibition by lysateﬁ'om ICI treated MCF-7 cells.
We have previously shown that ICI treated MCF-7 cells contain a factor capable of
inhibiting active Cdk4 in vitro, and that this factor is lost upon p21 immunodepletion
(59). We reasoned that the Cdk4-associated kinase activity present in ICI treated LCC9
cells could be due to the fact that the Cdk4 complex had become refractory to inhibition.
To test this possibility, Cdk4 activity in E2 treated MCF-7 and LCC9 cells was compared

in a lysate mixing assay.

112

A' ICI MCF7 ICI MCF7

BEL M + E2 MCF7 + E2 LCC9
MCF7 LCC9 MCF7 LCC9 . .
ICI E2 10 E2 10 E2 ICI E2 "“X W

 

 

 

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El LCC9
— El theoretical

 

 

 

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E2 1t05 1t02 1 to 1 ICI
mixing ratio

Figure 4: Cdk4 activity in LCC9 is sensitive to the inhibitory factor present in [CI
treated MCF-7 cells. Parallel cultures of MCF-7 and LCC9 cells were treated for 48 h in
ICI and E2 as described, and harvested. Cellular lysates were prepared and quantitated.
A) Representative autoradiogram showing Cdk4 inhbition in MCF-7 and LCC9 cells. In
the ﬁrst eight lanes, equal amounts of the indicated lysates were immunoprecipitated with
Cdk4 or control (mock) antibodies. The pellets were then incubated with radiolabeled
ATP and substrate (GST-Rb) and the reaction products resolved by SDS-PAGE. In the
ﬁnal six lanes, increasing amounts of lysate from ICI treated MCF-7 cells (20 ug, 50 ug
and 100 ug) were added to ﬁxed amounts of lysates from E2 treated MCF-7 or LCC9
cells (100 ug) as indcated, and the mixed lysates were incubated prior to
immunoprecipitation with Cdk4 antibodies and kinase reaction. B) The lysate mixing
experiment was repeated in triplicate and product bands were quantitated by
phosphorimaging. The graph shows the avereage Cdk4 activity in mixed and unmixed
lysates as a fraction of the activity recovered from lysates from E2 treated cells; error
bars indicate one standard deviation. The white bars ("theoretical") indicate the fractional
representation of E2 lysate in the mixtures at each mixing ratio (see text).

 

113

Cells were cultured in 100 nM ICI or E2 for 48 h, then harvested and cellular
lysates prepared. Increasing amounts (20, 50 and 100 ug) of ICI treated MCF-7 cell
lysates were added to a ﬁxed amount (100 ug) of E2 treated MCF-7 or LCC9 lysate. The
mixed lysates and unmixed controls were then incubated brieﬂy and subjected to Cdk4
kinase assay as described in Material and Methods. A representative autoradiogram is
shown in Figure 4A. Cdk4 activity in unmixed lysates is shown in the ﬁrst four lanes,
and kinase activity obtained with pre-immune control antibodies in the next four lanes.
Consistent with the results in Figure 2, Cdk4 associated kinase activity is tightly
regulated by ICI and E2 in MCF-7 cells. In LCC9 cells Cdk4 is active under both
conditions, and the activity is approximately 50% of that in E2 treated MCF-7 cells.

The Cdk4 activity recovered from the mixed lysates is shown in the last six lanes.
Consistent with our earlier report, Cdk4 in lysates of E2 treated MCF-7 cells was
inhibited in a dose dependent manner by the lysate from ICI treated MCF-7 cells. The
Cdk4 activity from LCC9 cells was not refractory to inhibition by lysate from ICI treated
MCF-7 cells. This experiment was performed three times, and the combined results are
shown in Figure 4B. The Cdk4 associated Rb kinase activity in the mixed lysates is
expressed relative to the activity in the unmixed lysate from both E2 treated samples.
The bars marked “theoretical” indicate the fractional representation of the E2 treated
lysate in each mixed lysate; for example at the 1 to 2 ratio, there is 100 ug of E2 treated
lysate and 50 ug of ICI treated lysate; the fractional representation of the E2 treated lysate
is 100/(100+50) = 0.67. At each mixing ratio, the Cdk4 activity recovered is signiﬁcantly
less than that predicted by dilution of the active enzyme with inactive enzyme, and there

was no signiﬁcant difference between the Cdk4 activity recovered from MCF-7 and

114

LCC9 cells. These data suggest that although Cdk4 is not inactivated by ICI in LCC9
cells, LCC9’s Cdk4 complexes remains sensitive to inhibition by the ICI regulated
inhibitory factor present in MCF-7 cells; this inhibitory activity is attributed to p21.

To support the generality of this ﬁnding, we transfected MCF-7 and LCC9 cells
with an expression vector encoding p21 as a fusion protein with green ﬂuorescent protein
(GFP) and an empty GFP control vector. Cells were then stained with a cell permeable
DNA binding ﬂuorescent dye, and cells were analyzed by dual parameter ﬂow cytometry
for GFP expression and DNA content. Proliferation in the presence of E2 was inhibited
by expression of GFP-p21 in both MCF-7 and LCC9 cell lines (data not shown),
suggesting that Cdk4 and/or Cdk2 in LCC9 cells remains sensitive to p21 inhibition, and
that this inhibition leads to cell cycle arrest.

One possible explanation for the constitutive Cdk2 and Cdk4 activity in LCC9
cells despite high p21 levels and sensitivity to inhibition by p21 is that LCC9 cells’ p21 is
mutant. A mutant p21 that is unable to inhibit Cdk activity while retaining Cdk binding
activity was previously isolated from primary human breast cancer (2). To determine if
this or some other mutation was responsible for LCC9’s lack of Cdk regulation, we
ampliﬁed the p21 and p27 coding sequences from MCF-7 and LCC9 cells by reverse
transcriptase-polymerase chain reaction using primers directed at their 3’ and 5’
untranslated regions. The entire coding sequences were sequenced, and no differences
existed between MCF-7 and LCC9. The p21 and p27 sequences from both cell lines
were identical to the published sequences (GenBank accession numbers AF497972 and
AF247551, respectively, data not shown). We therefore conclude that a mutant Cdkl

could not account for LCC9’s antiestrogen resistant phenotype.

115

MCF7 5523
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cyclin Di ‘_}

saw, 2» .uvir.

 
  

 

actin 1“ 1~ ~ the

Figure 5: LCC9 cells have elevated cyclin D levels and deregulated p21. MCF-7
and LCC9 were plated in FBS, then treated with either 10 nM ICI or 10 nM E2 for
48 h. Lysates were prepared, and quantitated, and aliquots were Western blotted

with antibodies recogniziung cyclin D1, cyclin B, Cdk2, p21, p27 and actin.

116

Altered expression of cell cycle regulators in LC C 9 cells. The altered regulation of Cdk4
and Cdk2 activity in LCC9 cells by ICI could be caused by changes in expression levels
of proteins which regulate Cdk activity. We compared the expression of a panel of cell
cycle regulators in MCF-7 and LCC9 cells in the presence of ICI and E2. Cells were
treated for 48 h with ICI or E2, then harvested and analyzed by Western blotting for
cyclins D, E and A, Cdk2, p21, p27 and actin. The results from one experiment are
shown in Figure 5.

Consistent with our earlier observation (Figure 2C), cyclin D protein levels were
slightly higher in LCC9 cells than in MCF-7 cells. Cyclin E and Cdk2 were expressed at
equal levels and were unregulated in the two cell lines. While expression of the Cdkls
p21 and p27 was upregulated after two day ICI treatment of MCF-7 cells, in LCC9 cells
p21 was not regulated and p27 was only slightly upregulated by ICI. In both ICI and E2
treated LCC9 cells, Cdkl levels were higher than in E2 treated MCF-7 cells. This

experiment was repeated with identical results.

Discussion

This work describes an analysis of the LCC9 cell line, an antiestrogen resistant
derivative of the MCF-7 human breast cancer cell line originally selected and
characterized by Brunner et a1 (6). Despite expressing ER, LCC9 exhibit E2 independent
and ICI resistant grth in vitro and in nude mice. However, the molecular basis for

antiestrogen resistance in this cell line is unknown. Here we compare regulation through

117

the G1 phase cell cycle check point in ICI and E2 treated LCC9 cells with the well
characterized responses of MCF-7.

As is the case in MCF-7 cells, a canonical ERE is transcriptionally activated by
E2 in LCC9 cells and this activation is inhibited by ICI (Figure 2). This suggests that
LCC9’s ER functions normally, although this conclusion must be qualiﬁed in two ways.
First, basal transcription from an ERE containing promoter, in the presence of ICI or in
CSS alone, is slightly but reproducibly higher in LCC9 cells than in MCF-7. It is
difﬁcult to judge the contribution of this small difference towards LCC9’s antiestrogen
resistance, but it is possible that the threshold of transcriptional activity required for
proliferation is quite low and LCC9 cells have enough ER activity in ICI to permit
growth. Second, this assay only measures transcriptional activity at a consensus ERE.
Growing evidence suggests that ER can regulate transcription via many different
promoter elements, including non-consensus EREs and ERE half sites (29, 37, 38, 49)
and AP-l sites (30, 39, 45). LCC9’s antiestrogen resistance may be due to an alteration
in ER mediated transcription of one or more critical genes, although the ER retains wild
type activity at a consensus ERE. Future studies will compare the regulation of
transcription at alternative ER regulated promoters in MCF-7 and LCC9 cells.

Transit through G] into S phase is dependent on the activity of both Cdk4 and
Cdk2, and these activities are regulated by posttranslational modiﬁcation, by association
with cyclins, and by association with Cdkls (27, 28, 35, 57). The proliferative potential
of a cell in any given context depends on a balance between activators (cyclins) and
inhibitors (i.e. p21) of G1 phase Cdks. In MCF-7 cells Cdk4 and Cdk2 are tightly

regulated by ICI and E2. In contrast, LCC9 cells’ G1 Cdk activities are not affected by

118

ICI or E2 treatment, although they remain sensitive to an inhibitory activity in ICI treated
MCF-7 cells previously attributed to p21 (Figures 3 and 4) (59). Both cyclin D1 and p21
appear to be expressed at higher levels in E2 treated LCC9 compared to MCF-7 cells, and
while in MCF-7 cells p21 is highly induced by two days treatment with ICI, p21 is not
regulated by ICI in LCC9 cells (Figure 5).

A cell in G1 phase decides whether or not to enter S phase based on the balance
of Cdk activators and inhibitors. In the presence of E2, the balance of activators and
inhibitors favors active Cdks in both MCF-7 and LCC9 cells, and active Cdks
phosphorylate Rb and other critical S phase substrates. In MCF-7 cells, ICI treatment
increases Cdkl levels until all Cdk4 and Cdk2 is inactive, and proliferation ceases. In
contrast, the balance remains in favor of active Cdks in ICI treated LCC9 cells due to a
lack of p21 upregulation, allowing antiestrogen resistant proliferation.

This work supports a model of acquired antiestrogen resistance in which signaling
alterations occur upstream of G1 Cdk activation. Both the increased cyclin D1
expression and the lack of p21 induction upon ICI treatment may contribute to active G1
Cdks, and ultimately proliferation. This points to a number of signal transduction
pathways that merit further study for their potential contribution to antiestrogen
resistance in LCC9 cells. During the preparation of this manuscript, Clarke et al, using
expression and promoter analysis, reported that levels of transcription from a cAMP
response element was increased in LCC9 cells compared with their parental cell line, and
that levels of epidermal growth factor receptor (EGF-R), which activates STATS, were
lower (21). These reported alterations may account for elevated levels of cyclin D1 and

the lack of p21 regulation observed in LCC9 cells. Estrogen regulation of the cyclin D1

119

promoter has been demonstrated to involve a CAMP response element (CRE), (8, 55) and
the induction of p21 is dependent upon STAT signaling (3, l2). Overactive CRE
transcription could lead to increased cyclin D] levels, and decreased levels of EGF-R
could depress STAT signaling and account for the lack of p21 regulation in LCC9 cells.
Further comparison of these signaling pathways in MCF-7 and LCC9 cells to determine
if corruption of one or more pathways could contribute to the development of acquired

antiestrogen resistance is warranted.

120

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127

CHAPTER FIVE

Conclusion

128

Chapter 5: Conclusion

Introduction. Antiestrogens are drugs that bind to ERa’s ligand binding domain and
prevent transcription of some or all E2 regulated genes. Because of E2’s role as a
mitogen in normal and malignant breast tissue, antiestrogens such as tamoxifen have
been used to successfully treat breast cancer patients with ERa positive tumors since the
1970s (19, 21). One limitation of their use is that during treatment, many patients’
tumors bypass their requirement for E2 and become insensitive to growth arrest by
antiestrogens (35). Despite much attention, the molecular mechanisms responsible for
this change in phenotype is not well understood. Research in this area may lead to better
therapeutic techniques that avoid or delay the problem of antiestrogen resistance.

In hormone sensitive cells, E2 promotes passage through the G1 phase of the cell
cycle, and antiestrogens cause a G1 phase cell cycle arrest (32). The G198 phase
transition is controlled by the sequential activation of Cdk4 and Cdk2, which
phosphorylate Rb; Cdk2 also has additional substrates. Cdk activity depends on
association with a cyclin, a regulatory subunit. Cdk activity can be further modiﬁed by
association with Cdkls, such as p21 (16, 17, 29). In clinical breast cancer, the main
regulatory subunit of Cdk4, cyclin D1 , is often overexpressed, and overexpression
correlates with ERa-positive status (15, 18, 27). Investigations into hormone signaling in
models of human breast cancer revealed that E2 activates, and antiestrogens inhibit, Cdk4
and Cdk2 activities. It is proposed that E2 increases cyclin D1 protein levels, which
activates Cdk4 and provides additional binding targets for p21, tritrating it from
inhibitory association with Cdk2 (24). E2 also decreases p21 protein levels, contributing

to further increases in Cdk activity.

129

These observations led us to investigate the possible relationship between the
regulation of G1 phase Cdk activity and antiestrogen resistance. The hypothesis of this
dissertation is that alterations in signaling pathways leading to inappropriate activation of
G1 Cdks contributes to antiestrogen resistant proliferation in in vitro models of human
breast cancer cells. This hypothesis was explored by examining the regulation of Cdk4
by E2 and the pure steroidal antiestrogen ICI in the E2 dependent, ICI sensitive MCF-7

cell line (chapter 2). We then studied the effects of expressing ectopic cyclin D1 on

nsIA's-I“. um

regulation of Cdks and proliferation in an MCF-7 subclone engineered to conditionally

express cyclin D1 (chapter 3). Finally, we compared the effects of E2 and ICI on MCF-7

 

with LCC9 cells, an ICI resistant derivative of MCF-7 (chapter 4).

1E.

Signiﬁcance of major ﬁndings. In chapter 2, the regulation of Cdk4 by ICI and E2 in
MCF-7 cells was examined. In time course experiments, Cdk4 activity was inhibited by
ICI and activated by E2 treatment, consistent with previous reports (8, 26, 33). In
contrast to previous reports, no correlation was observed between levels of cyclin D1
protein and Cdk4 activity, although there was an inverse correlation between p21 protein
levels and Cdk4 activity. Through lysate mixing experiments, we demonstrated that
lysates of ICI treated MCF-7 cells inhibit active Cdk4 from E2 treated MCF-7 cells in a
dose dependent manner. Furthermore, immunodepletion of p21, but not p27, from the
lysate of ICI treated cells abolished the Cdk4 inhibitory activity in the lysate, suggesting
p21 was responsible for Cdk4 inhibition. Although p21 may serve as an activator of
cyclin Dl-Cdk4 complexes under some conditions (4, 30, 34), in the context of ICI

mediated grth arrest it clearly acts as a Cdk4 inhibitor.

130

We conclude that p21 is an ICI regulated inhibitor of Cdk4 that can act
independently of changes in Cdk4’s major regulatory subunit, cyclin D1. These results
corroborate the work of Foster et al, who showed that E2 downregulates Cdkl expression
in MCF-7 cells even when cell cycle arrest is enforced inhibition of cyclin Dl-Cdk4 by
ectopic p16, suggesting that E2 directly regulates Cdkl expression independently of
progression through the cell cycle (7). In MCF-7 cells, p21 controls the activity of
critical G1 -)S phase regulators, and is a direct target of ICI and E2.

The inhibitory activity of p21 can be overcome by ectopic expression of Cdk4’s
main regulatory subunit, cyclin D1. MCF-7 cells were engineered to conditionally
express epitope tagged human cyclin D1 to assess its potential to contribute to acquired
antiestrogen resistance (chapter 3). Consistent with results from other labs, we found that
ectopic cyclin D1 expression caused G1 arrested cells to enter S phase in the presence of
antiestrogen, but was insufﬁcient to support long term antiestrogen resistant proliferation.
In time course experiments, ectopic cyclin D1 activated Cdk4 in the presence of ICI.
While E2 treatment resulted in sustained Cdk4 activation, expression of ectopic cyclin
D1 in the presence of ICI did not sustain activity beyond 72 h, although ectopic cyclin D1
remained in complex with Cdk4 up to 120 h. Levels of p21 protein increased during
antiestrogen treatment, and decreased with E2 treatment.

These data provide a possible mechanism for the paradoxical ability of ectopic
cyclin D1 to cause ICI arrested cells to enter the cell cycle, but its inability to promote
long term antiestrogen resistant proliferation (20, 22, 36). We propose a model whereby
in ICI arrested cells, Cdk4 is in excess and the amount of cyclin D1 is limiting for

proliferation because all cyclin Dl/Cdk4 complexes are in inhibitory association with a

131

Cdkl. Increasing the amount of cyclin D1 using regulated expression systems transiently
increases the amount of p21-free cyclin D1/Cdk4 complexes, which are active kinases
and promote proliferation. Upon long term treatment with ICI, p21 levels accumulate
such that all cyclin D1-Cdk4 complexes contain p21 and are catalytically inhibited.
When cells are treated with ICI, p21 levels may increase up to some ﬁxed level, or,
alternatively, cells may have a “sensor” of Cdk4 activity which acts with ICI to increase
p21 levels until all cyclin Dl-Cdk4 complexes are inhibited. In support of this

possibility, in other systems expression of ectopic cyclin D1 has been shown to increase

("‘12 3 _'I

E2F transcription factor binding and activity at the p21 promoter, leading to increased

 

p21 protein levels (13, 14). This model predicts that cyclin D1 ampliﬁcation alone is an

F|—_.,, 9, .. ..

unlikely cause of acquired antiestrogen resistance, and that additional changes, such as in
Cdkl regulation of Cdk complexes, would be necessary for the development of
antiestrogen resistant tumors.

To further examine potential changes in G1 -)S phase regulation that may
contribute to antiestrogen resistance, the MCF-7 cells were compared with the LCC9 cell
line, an MCF-7 derivative selected for ICI resistant growth (chapter 4) (2). LCC9 cells
were insensitive to growth arrest by ICI, despite having an apparently normally
functioning ER. While Cdk4 and Cdk2 are inactivated following ICI treatment of MCF-7
cells, in LCC9 both Cdks remain active. Active Cdk4 from LCC9 cells was not
refractory to inhibition by p21 from lysates prepared from ICI treated MCF-7 cells,
suggesting that no change in the Cdk4 complex’s ability to be inhibited by p21 occurred
during selection of the LCC9 cell line. However, while levels of p21 and p27 in MCF-7

cells increased after two days ICI treatment, p21 was not regulated by E2 and ICI in

132

LCC9 and p27 was only slightly increased by ICI. In addition, while cyclin D1 protein
levels were not regulated by ICI and E2 in either cell line, LCC9 cells showed
consistently higher levels of cyclin D1 than the parental MCF-7 cells.

In LCC9 cells, increased cyclin D1 levels may increase the total amount of cyclin
D1-Cdk4 complexes, and deregulated Cdkls may lead to constitutively active Cdk4 and

Cdk2. The unregulated Cdks would lead to increased phosphorylation of Rb and Cdk

substrates, contributing to estrogen insensitive and antiestrogen resistant grth (Figure r
1). However, the fundamental causes of the changes in cyclin D1 levels and p21 .
regulation in the LCC9 cell line are not known.

E2 is reported to regulate the cyclin D1 promoter by ERa’s direct interaction with is.

 

Spl at Spl binding sites, and indirectly by protein kinase A dependent and independent
CAMP response element activation (CRE) (3, 28); cyclin D1 transcription is also
regulated positively by NF-KB (12). Transcription of p21 is activated by Spl and STAT
transcription factors, and Myc association with Spl and Sp3 inhibits p21 expression; Myc
levels are increased by E2 treatment in breast cancer cells. (5, 6, 9, 31). STATS are
activated by a number of cytokine and tyrosine kinase receptors and are implicated in p21
mediated growth arrest by induced by interferon-y and transforming growth factor-[3 (5,
10). STAT induced expression of p21 is also implicated in the growth arrest caused by
epidermal growth factor (EGF) in the MDA468 human breast cancer cell line, which
overexpresses EGF-receptor (EGF-R).

The transcriptional regulation of cyclin D1 and/or p21 may underlie LCC9s
antiestrogen resistant phenotype. One point at which cyclin D1 and p21 regulation by ER

ligands may intersect is the Spl transcription factor. Both promoters are activated by Spl

133

(3, 9), and direct interaction between ER and Spl is responsible for E2 mediated
transcription of progesterone receptor (23). Myc acts as a transcriptional repressor at Spl
sites of the p21 promoter (9). If Myc expression or its activity was increased through
mutation or epigenetic change, the induction of p21 by ICI might be diminished or
abolished. Myc levels are increased by E2 and decreased by ICI, and ectopic Myc
expression in MCF-7 cells has been reported to cause ICI arrested cells to enter the cell
cycle and activates cyclin E-Cdk2 without activating cyclin D1-Cdk4 (6, 25), suggesting
that Myc may act downstream of cyclin D1.

Other alterations in the regulation of cyclin D1 and p21 might also be involved in
causing the LCC9 cell line’s antiestrogen resistance. MCF-7 cells’ basal and ICI
regulated gene expression was recently compared with LCC9s (11). LCC9 cells have
increased levels of basal transcription from CRE and NF-IcB site reporter plasmids, and
unlike MCF-7 cells, their NF-KB activity is not inhibited by ICI. The cyclin D1 promoter
is controlled by both a CRE and NF-KB binding site (12, 28), and increases in their
activity may account for the increased level of cyclin D1 protein observed in LCC9 cells
compared to MCF-7 cells. In addition, LCC9 cells are reported to have decreased levels
of EGF-R expression and protein (11). EGF-R activates STAT transcription factors,
which are implicated in p21’s transcriptional regulation (1 , 5). The reduced levels of
EGF-R in LCC9 compared to MCF-7 cells may result in reduced STAT activation and
reduced or deregulated expression of p21. Further examination of these signaling

pathways for their potential contribution to acquired antiestrogen resistance is warranted.

134

 

NF-ch Cdk4 Cdk2 —> ""°”"°”'a“°"°'

S phase substrates
Rb

CYClln D1 EZF regulated
ﬂ gene transcription

I '. E2F /

8p1 CRE

O
.0
O
0

E2

Figure 1: Alterations in regulation of GI Cdks may lead to antiestrogen resistance.
In hormone sensitive human breast cancer cells, proliferation is controlled by the
balance between activating (cyclin D1) and inhibitory (p21) factors which regulate the
activity of Cdk4 and Cdk2. E2 increases the level of cyclin D1 by activating
transcription at Spl and CRE sites in the cyclin D1 promoter; cyclin D1 transcription
is also regulated by NF-KB. E2 decreases levels of Cdkls such as p21, although
mechanisms of transcriptional regulation are not described. In growth arrest by
cytokine and receptor tyrosine kinases, p21 is upregulated by Spl and STAT
transcription factors, and p21 is expression is inhibited by Myc. In the antiestrogen
resistant LCC9 cell line, cyclin D1 levels are higher than their parental MCF-7 cells,
and p21 protein levels are not regulated by ICI. Points at which the regulation of
cyclin D1 and p21 may be altered in antiestrogen resistant LCC9 cells are shown with
dashed lines.

135

Model of E2 and 1C1 regulation of GI 9S in MCF-7 and implications an acquired
antiestrogen resistance. The experiments presented in the preceding chapters tested the
hypothesis that inappropriate activation of Cdk4 and Cdk2 contributes to antiestrogen
resistant proliferation in an in vitro model of human breast cancer. In addition to being
an inhibitor of Cdk2, we have demonstrated that p21 is an ICI regulated inhibitor of
Cdk4, and that its expression is deregulated in the LCC9 cell line, an antiestrogen
resistant variant of the MCF-7 cell line. Furthermore, ectopic expression of cyclin D1
causes ICI arrested cells to enter S phase, and cyclin D1 levels are higher in LCC9 than
MCF-7 cells. The data support the hypothesis, with the following qualiﬁcations:

1) The expression of ectopic cyclin D1 did not support long term Cdk4 activation
or proliferation, suggesting that this single change contributes towards, but is unlikely to
cause, antiestrogen resistance. In this regard, LCC9 cells, selected from the MCF-7 cell
line in multiple steps, have multiple alterations, including elevated cyclin D1 protein
levels and p21 deregulation.

2) Although the regulation of LCC9 cells’ Cdk activity is dramatically different
than in MCF-7 cells, the composition and basic function of Cdk4 complexes appears
normal. This suggests that the cause of Cdk deregulation is not due to mutational change
of a Cdk complex member, but that the ultimate causes are likely to be changes in
upstream regulators, particularly cyclin D1 and p21.

These experiments support a model in which breast cancer cell proliferation is
determined by balance between positive factors, e.g. cyclin D1, and negative factors, e.g.
p21, which regulate the activity of G1 phase Cdks (Figure 1). In antiestrogen sensitive

cells, antiestrogen shifts the balance in favor of negative factors, inhibiting G1 Cdk

136

 

activity and ultimately proliferation. In antiestrogen resistant cells, deregulated
expression of cyclins and Cdkls maintains the balance in favor of active G1 Cdks,
allowing proliferation in the presence of antiestrogen. Multiple molecular changes
upstream of cyclin and Cdkl expression may contribute to antiestrogen resistance in

human breast cancer.

137

 

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