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Targeting molecular signals involved in the development of castration resistant prostate cancer

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Targeting molecular signals involved in the development of castration resistant prostate cancer

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Content TARGETING MOLECULAR SIGNALS INVOLVED IN THE DEVELOPMENT
OF CASTRATION RESISTANT PROSTATE CANCER

by

Llana Pootrakul

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PATHOBIOLOGY)

December 2007

Copyright 2007 Llana Pootrakul

ii
DEDICATION

To my family, Dad, Mom, Jamie, and Shane, who have always given me
unconditional love and support.

iii
ACKNOWLEDGEMENTS

First, I would like to express my deep and sincere gratitude to my mentor, Dr.
Richard J. Cote, whose drive and enthusiasm inspire me. His support and guidance
have truly been invaluable to me, and this work could not have been accomplished
without him.

This research project also would not have been possible without the support of the
members of my thesis committee. I would like to thank Dr. Ram Datar, who has
encouraged me to strive for greater scientific knowledge. I am very thankful to Dr.
Louis Dubeau, who has been an exceptional teacher and advisor to me for many
years. Deepest gratitude is also expressed to Dr. Gerhard Coetzee, whose brilliant
advice and support have contributed greatly to this thesis.

I would also like to express my gratitude to Dr. Clive R. Taylor, Dr. Shan-Rong Shi,
Dr. Mohammad Alavi, and Dr. Yan Shi who have been abundantly helpful and
offered extraordinary guidance, and without whose knowledge these studies would
not have been successful.

I am also grateful to Dr. Amy S. Lee and Dr. Michael Press who have provided
valuable advice and contributed greatly to this project.

iv
Special thanks goes to the members of the Cote Lab who have taught, helped,
listened, and worked with me, and without whom this thesis would not be complete,
including Lillian Young, Carmela Villajin-Busque, William Win, Marija Balic,
Cheng Liu, Debra Hawes, Henry Lin, Anthony Williams, Mark Birkhaun, Stephen
Beil, Laurie Tang, and Anirban Mitra.

I would also like to convey thanks to the Department of Pathology at USC Keck
School of Medicine for the invaluable assistance of the administration and constant
support from faculty and fellow students.

Finally, I wish to express my love and gratitude to my family, for their
understanding, love, and never-ending support throughout the duration of my studies.

v
TABLE OF CONTENTS

Dedication………………………………………………………………………….ii

Acknowledgements………………………………………………………………..iii

List of
Tables……………………………………………………………………………....vii

List of
Figures……………………………………………………………………………..viii

Abstract…………………………………………………………………………….x

Chapter One: Introduction
1.1 The Prostate Gland……………………………………………………………...1
1.2 Prostate Cancer
1.2.1 Epidemiology………………………………………………………….2
1.2.2 Prostate Cancer Staging……………………………………………….3
1.2.3 Hormone Dependence and Castration Resistance…………………….3
1.3 Stress Response Proteins
1.3.1 Overview of the Stress Response Protein Family………………….….15
1.3.2 The Unfolded Protein Response………………………………………16
1.3.3 Grp78………………………………………………………………….17
1.4 Apoptosis
1.4.1 Overview……………………………………………………………....19
1.4.2 Inhibitors of Apoptosis………………………………………………..20
1.4.3 Survivin………………………………………………………………..22
1.5 The Her-2/neu Pathway…………………………………………………………23
1.6 The Cell Cycle
1.6.1 Overview………………………………………………………………24
1.6.2 Cyclin D1……………………………………………………………...27
1.7 Recent Novel Findings in Prostate Cancer……………………………………...29
1.8 Purpose of Study………………………………………………………………...30

Chapter Two: Increased Grp78 Expression is Associated with Prostate
Cancer Progression and the Development of Castration Resistance
2.1 Introduction……………………………………………………………………..32
2.2 Materials and Methods………………………………………………………….34
2.3 Results…………………………………………………………………………..39
2.4 Discussion……………………………………………………………………….52

vi

Chapter Three: Expression and Cellular Localization of Survivin is
Associated with Prostate Cancer Aggressiveness, Clinical Outcome,
and the Development of Castration Resistance
3.1 Introduction………………………………………………………………..…..57
3.2 Materials and Methods………………………………………………………...59
3.3 Results…………………………………………………………………………64
3.4 Discussion…………………………………………………………………..….79

Chapter Four: Her-2/neu Promotes Prostate Cancer Cell Growth and
Survival and is Involved in Cyclin D1 Regulation
4.1 Introduction……………………………………………………………………83
4.2 Materials and Methods………………………………………………………...86
4.3 Results…………………………………………………………………………88
4.4 Discussion……………………………………………………………………...92

Chapter Five: Targeting Molecular Mechanisms Involved in Cell Growth,
Proliferation and Survival: Effect on Castration Resistant Cells
5.1 Introduction…………………………………………………………………....93
5.2 Materials and Methods……………………………………………….………..94
5.3 Results……………………………………………………………………….....97
5.4 Discussion……………………………………………………………………...114

Chapter Six: Conclusions and Impact…………………………………………..117
References…………………………………………………………………………122

vii
LIST OF TABLES

Table 2-1: Grp78 expression (immunoreactivity) in untreated T
3
N
0
M
0,
treated
T
3
N
0
M
0
, and castration resistant prostate cancer…………………………………...46

Table 2-2: Quantification of protein expression from western blot
analysis of Grp78………...………………………………………………………….48

Table 2-3: Grp78 expression and recurrence-free or overall survival of
patients with untreated stage T
3
N
0
M
0
tumors……………………………………….50

Table 3-1: Survivin cytoplasmic expression (immunoreactivity) in untreated
T
3
N
0
M
0
, treated T
3
N
0
M
0
, and castration resistant prostate cancer
(CRPC)……………………………………………………………………………...73

Table 3-2: Survivin nuclear expression (immunoreactivity) in untreated
T
3
N
0
M
0
, treated T
3
N
0
M
0
, and castration resistant prostate cancer (CRPC)……...74

Table 4-1: Correlation between ErbB-2 and Cyclin D1 immunostaining in clinical
specimens……………………………………………………………………………………91

viii
LIST OF FIGURES

Figure 1-1: Five proposed molecular pathways and signaling mechanisms in
the development of castration resistant prostate cancer………...…………………..7

Figure 1-2: Major pathways in apoptosis…………………………………………..21
Figure 2-1: Grp78 expression in prostate cancer……………………………………45
Figure 2-2: Grp78 expression in prostate cancer cells during brief and
prolonged androgen starvation……………………………………………….……..47

Figure 2-3: Probability of recurrence-free (clinical and/or PSA) status in 164
patients with stage T
3
N
0
M
0
prostate cancer, based on levels of Grp78
immunoreactivity……………………………………………………………………49

Figure 2-4: Probability of recurrence-free (clinical and/or PSA) status in 80
patients with stage T
3
N
0
M
0
prostate cancer stratified by median age, based on
levels of Grp78 immunoreactivity…………………………………………………..51

Figure 3-1: Survivin expression in prostate cancer………………………………....72

Figure 3-2: Survivin expression in prostate cancer cell lines……………………….75

Figure 3-3: Survivin localization in prostate cancer cells upon androgen
deprivation and castration resistant growth……………..…………………………..76

Figure 3-4: Probability of recurrence-free (clinical and/or PSA) status in 122
patients with stage T
3
N
0
M
0
prostate cancer, based on combined intensity and
percent of tumor cells (status) with nuclear Survivin immunoreactivity…………...77

Figure 3-5: Probability of recurrence-free (clinical and/or PSA) status in 122
patients with stage T
3
N
0
M
0
prostate cancer, based on percent of tumor cells
with nuclear Survivin immunoreactivity……………………………………………78

Figure 4-1: Cyclin D1 expression in prostate cancer……………………………….90

Figure 5-1: Serial dilution of Grp78 and Survivin siRNA transfection…………...101

Figure 5-2: Comparison of effects of control siRNA and lipofectamine levels
on apoptosis………………………………………………………………………..106

ix

Figure 5-3: Western blot analysis of induction of apoptosis following
combined targeted treatment in castration resistant C42B cells…………………...107

Figure 5-4: Fluorescence analysis of apoptosis induction through membrane
expression of annexin V…………………………………………………………...110

Figure 6-1: Potential Therapeutic Targets…………………………………………119
Figure 6-2: Targeting Basic Hallmarks of Cancer…………………………………120

x
Abstract

A significant clinical problem is that patients who are initially responsive to
androgen ablation therapy often develop castration resistant carcinoma of the
prostate (CRPC), despite recent advances in cancer therapy. A better understanding
of the complex contributing molecular mechanisms underlying the development of
CRPC is necessary so that the most appropriate therapeutic targets may be identified.
In order to further our understanding of the development of castration resistance, we
have established a unique clinical cohort designed to represent successive stages in
the development of CRPC. Immunohistochemical analysis (IHC) on clinical
specimens allows for identification of potential prognostic markers of prostate
cancer, and the use of in vitro cell line models allows for molecular manipulation and
assessment of signal transduction events in the progression to CRPC, and will also
permit testing of novel rational therapeutics. Our lab has previously identified that
overexpression of receptor tyrosine kinase Her-2/neu plays a significant role in
castration resistant cell proliferation and survival [Shi 2006]. Recently, involvement
of many additional proteins in the development of castration resistance has been
suggested, including induction of survival-associated proteins Survivin and Grp78
(78 kDa glucose-regulated protein). Survivin has been shown to promote resistance
to antiandrogen treatment in prostate cancer, both in vitro and in vivo [Fortugno
2002, Zhang 2005]. Immunoreactivity of stress response protein Grp78 has been
identified as a putative marker of CRPC [Mintz 2003],

while preclinical data has
validated Grp78 as a functional cell surface molecular target for prostate cancer
xi
[Arap 2004]. This study identifies both Grp78 and Survivin as novel survival-
associated markers involved in the development of castration resistant prostate
cancer, and directly examines the combined roles of Grp78, Survivin, and Her-2/neu
in cell survival during the development of castration resistance. This is
accomplished by downregulation of Survivin and/or Grp78 expression through
siRNA interference, and blockage of Her-2/neu activity via Herceptin treatment in
castration resistant C42B cells. It is the hope that these experiments will provide
valuable information as to whether Grp78, Survivin, and Her-2/neu can form a
potentially therapeutic combination of targets for the management of CRPC.

1
Chapter One: Introduction

1.1 The Prostate Gland
The prostate gland is a male accessory sex organ that aids in the formation of
semen during ejaculation. The prostate lies just inferior to the bladder, surrounds the
posterior urethra, and rests on the genitourinary diaphragm. The normal prostate
weighs approximately 20-30g and is composed of tubuloalveolar glands arranged in
lobules and fibromuscular stroma surrounding the glands. As stated by McNeal
[McNeal 1998], the prostate gland is divided into three distinct zones: peripheral
zone, central zone, and transitional zone. The peripheral zone is the largest of the
three, lies posterior and laterally in the prostate, and is the zone that is predominantly
affected by prostate cancer. The central zone is the second largest, and surrounds the
ejaculatory ducts as they extend from the base of the prostate to the verumontanum.
The transitional zone makes up a small portion of the prostate volume and surrounds
the prostatic urethra.
Microscopically, the prostate gland is composed mainly of two cell types and
fibromuscular stroma. First are the luminal glandular cells, which are columnar or
cuboidal shaped. Luminal glandular cells are terminally differentiated and secrete
prostate specific antigen (PSA), prostatic acid phosphatase (PAP), and additional
enzymes. These androgen dependent cells express the androgen receptor, and are
thus regulated by androgen (principally testosterone and dihydrotestosterone [DHT]).
Basal cells are flat and are arranged at the periphery of prostate glands, between the
secretory cells and the basement membrane. These cells do not have secretory
2
function but are thought to represent the stem cell population within the prostate
[Tanagho 2003, McNeal 1998, Mills 2004].

1.2 Prostate Cancer
1.2.1 Epidemiology
Other than skin cancers, prostate cancer is the most prevalent cancer in
American men. It has been estimated by the American Cancer Society (ACS) that
approximately 218,890 new cases of prostate cancer will be diagnosed in the United
States in 2007. Prostate cancer rarely manifests before the age of 40, but the risk
rises dramatically thereafter [Crum 1994]. Roughly 1 in every 6 men will be
diagnosed with prostate cancer during his lifetime, but death occurs in only 1 in 34
men. While over 1.8 million men in the United States are survivors, prostate cancer
is still the second leading cause of cancer death in American men, behind lung
cancer. It has been estimated for 2007 that 27,050 men in the United States will die
of prostate cancer, which currently accounts for about 10% of cancer-related deaths
in men [www.cancer.org]. Prostate cancer may be a relatively benign condition for
some patients, but identification of etiology, appropriate management, and effective
therapies for this disease are still clearly major concerns among researchers and
clinicians.

3
1.2.2 Staging
The most widely used classification to describe the extent of cancer
progression is the Tumor, Nodes, Metastasis (TNM) System. According to the TNM
Classification of Malignant Tumours, T describes the primary tumor, N is the state of
regional lymph nodes, and M characterizes distant metastasis. The TNM clinical
staging of prostate cancer is outlined by stages T1-T4, where stage T4 represents the
furthest stage of progression. In stage T1, the tumor is not clinically apparent and is
not palpable or visible by imaging. The tumor has enlarged in stage T2, but remains
confined to the prostate. Stage T3 is characterized by extension of the tumor through
the prostatic capsule, including invasion of the seminal vesicles. By stage T4, the
prostate tumor is fixed or has invaded adjacent structures other than the seminal
vesicles. Additional sub-stages of prostate cancer in all stages T1-T4 distinguish
cancer progression within the stages [UICC]. Identification of the correct prostate
cancer stage is crucial for determining the most appropriate prostate cancer treatment
for a particular patient.

1.2.3 Hormone Dependence and Castration Resistance
The prostate gland is regulated by the actions of androgens on the androgen
receptor (AR). This activity promotes normal development, growth, differentiation,
and the function of the prostate [Partin 1998]. Testosterone, the major androgen in
the peripheral circulation, is synthesized primarily by the testes and is also secreted
by the adrenal glands. Conversion of testosterone to more biologically potent
4
dihydrotestosterone (DHT) by enzyme 5α-reductase occurs after testosterone
passively diffuses into prostate cells. Androgen signaling is mediated through the
AR, which belongs to the superfamily of ligand-activated nuclear transcription
factors. The AR gene is can be identified on the q11-12 region of the long arm on
the X chromosome, and runs approximately 90-100kb. The AR protein has a
molecular weight of 100-110kDa and contains four major functional domains: the
carboxy-terminal ligand-binding domain, the hinge region, the central DNA binding
domain, and the amino-terminal transactivation domain [Jenster 1991]. When the
AR is not bound to ligand, it is localized in the cytoplasm and complexed with
chaperone proteins, including Hsp90. Androgen binding to the AR leads to a
conformational change, followed by AR release from chaperone proteins and AR
activation. The sequence of events which follows includes AR phosphorylation,
dimerization, nuclear localization, and binding of the AR to androgen responsive
elements (HRE) on the DNA of androgen-responsive genes. Transcription of
androgen-dependent genes is further regulated by AR via protein-protein interactions
with AR coregulators (coactivators/corepressors) [Chang 2002].
The growth and development of prostate cancer depends on the presence of
androgens for signaling in the initiation and progression of prostate cancer,
resembling the original prostate epithelium which requires the presence of testicular
androgens during development and puberty [Feldman 2001]. Thus, the mainstay of
treatment for advanced prostate cancer is androgen ablation therapy, based on the
observation by Huggins that androgen ablation therapy through orchiectomy induces
5
regression of androgen dependent prostate cancer in up to 80% of patients [Huggins
1941]. Anti-hormonal management options, in addition to surgical castration,
include administration of analogues of gonadotropin releasing hormone (GnRH) to
suppress testosterone levels, antiandrogens (flutamide, bicalutamide) that inhibit the
binding of androgen to its receptor, or combinations of GnRH analogues and
antiandrogens [Schroeder 1998]. Although prostate tumor regression occurs in
response to androgen ablation therapy, prostate cancer cells can acquire the ability to
survive under these conditions and proliferate under very low levels of androgen.
The major obstacle in treatment of advanced prostate cancer is that most of
the patients who initially respond to hormonal therapy will eventually develop
resistance to hormonal therapy, a condition for which there is currently no effective
treatment. Thus, understanding the molecular basis of the development of castration
resistance is key for the management of advanced prostate cancer and CRPC, and
still remains a significant problem in the field of prostate cancer research.

6
Figure 1-1. Five proposed molecular pathways and signaling mechanisms in the
development of castration resistant prostate cancer.

a. Hypersensitive AR. Enhancement of AR sensitivity involves mechanisms
resulting in increased AR expression or activity due to amplification of the AR gene
[Visakorpi 1995; Koivisto 1997], enhanced stability and nuclear localization of AR
[Gregory 2001] or increased availability of more potent form of testosterone (DHT)
in environment surrounding prostate cancer cells [Labrie 1986].

b. Promiscuous AR. Broadened specificity of AR, allowing binding and activation
by non-androgen ligands, may result from AR gene mutations, such as T877A or
L701H, or increased expression of coactivators such as SRC1, SRC2, or ARA70
[Miyamoto 1998; Gregory 2001].

c. Outlaw mechanism. Outlaw signaling involves ligand-independent activation of
the AR by non-androgenic growth factors, such as EGF or IGF, [Sherwood 1998;
Kaplan 1999; Nickerson 2001] or activation of AR via RTK signaling (Her-2/neu
overexpression) through downstream PI3K/Akt and/or MAP kinase pathways [Culig
1994; Craft 1999; Yeh 1999; Wen 2000].

d. Bypass of AR signaling. Increased survival anti-apoptotic signaling, via
mechanisms such as elevated Survivin expression, Bcl-2 expression, and increased
7
expression of stress response proteins (Grp78), may contribute to prostate cancer cell
escape from androgen dependence through mechanisms independent of
androgen/androgen receptor activation [Liu 1996; Gleave 1999].

e. Lurker principle. This mechanism requires the existence of castration-resistant
sub-clones among androgen-dependent prostate cancer cells, which obtain a selective
growth advantage over the androgen-dependent cells following androgen ablation
therapy [Isaacs 1999].

Figure 1-1.

*Adapted from Feldman and Feldman 2001
Survival signaling
8
As previously stated, development of resistance to androgen ablation therapy
and severe depletion of therapeutic options persist as the predominant challenges in
the management of advanced prostate cancer. Androgen dependent prostate cancer is
characterized by the ability of cancer cells to undergo apoptosis in response to
hormone ablation. The transition to castration resistant prostate cancer (CRPC)
requires the survival of tumor cells despite androgen ablation, which may be
attributed to molecular mechanisms resulting in increased cell proliferation,
dysregulation of cell growth, or evasion of apoptosis. There are five hypotheses that
have been proposed to explain how cells might develop castration resistance
[Feldman 2001; Figure 1-1]: 1) Androgen receptor (AR) with enhanced sensitivity;
2) AR promiscuity, where AR has broadened specificity; 3) ‘Outlaw’ activity,
allowing ligand-independent AR activation; 4) Androgen-AR bypass, by inhibition
of apoptosis and promotion of survival, where altered anti-apoptotic pathways
diminish androgen/AR dependence; and 5) “Lurker” existence, where an androgen-
insensitive sub-clone of prostate cancer cells acquires a selective growth advantage
due to elimination of hormone-sensitive cells following hormone ablation, as
previously described by Feldman et al. These mechanisms are further described in
detail below.

AR Dependent Signaling
Gregory et al observed that the concentration of dihydrotestosterone (DHT),
the more potent form of testosterone, required to stimulate growth in castration
9
resistant prostate cancer cells is four times lower than that required for androgen-
dependent cells, and that the AR remains transcriptionally active in CRPC and can
increase cell proliferation even with castrate levels of androgen [Gregory 2001].
Thus, the androgen receptor and its natural ligand remain significant factors in
prostate cancer progression, even in patients who have undergone androgen ablation
therapy.

Hypersensitive AR
The hypersensitive AR pathway to castration resistance depends on both the
presence of androgen, albeit at low levels, and the activity of its receptor. One
mechanism leading to AR hypersensitivity is AR gene amplification, which directly
increases the AR available to bind with androgen to maximize the effectiveness of
even low levels of androgen [Visakorpi 1995, Koivisto 1997]. Visakorpi et al
reported AR amplification in 30% of recurrent prostate tumors, which was not
observed in the same patients prior to anti-androgen therapy [Koivisto 1997].
Alternatively, enhanced AR stability and/or nuclear localization may also allow for
hypersensitive AR [Gregory 2001].
Enhanced transcription via steroid receptor coactivators also promotes AR
activity under castrate levels of androgen. Gregory et al have shown that
overexpression of coactivators TIF2 and SRC1 in prostate cancer cells transactivates
AR to enhance its response to low levels of androgen, and that SRC-1 and TIF-2 are
increased in recurrent prostate cancer as compared to benign prostatic hyperplasia or
10
androgen dependent prostate cancer [Gregory 2001]. A role for AR coactivator AIB-
1 in prostate cancer cell growth and survival has also been demonstrated [Zhou
2005], and AIB-1 expression has been correlated with poorer clinical outcome in
prostate cancer [Gnanapragasam 2001]. Forced expression of the transcriptional
coactivator coactivator-associated arginine methyltransferase 1 (CARM1) has also
been shown to stimulate AR transcriptional activity, where siRNA knockdown of
CARM1 induces apoptosis and results in inhibited prostate cancer cell proliferation
[Majumder 2006]. Clinical studies have shown that expression of CARM1 in CRPC
tumors is significantly increased as compared to tumors from patients who had not
previously undergone hormonal therapy [Hong 2004, Majumder 2006].
In addition, increased local availability of DHT to prostate cancer cells may
also contribute to AR activation via the hypersensitive pathway [LaBrie 1986]. It
has been shown that prostate cancer cells are able to convert adrenal androgens to
DHT, and while serum testosterone has been shown to decrease by 95% following
androgen ablation, serum DHT in the prostate decreases by only 60% [Koh 2001,
LaBrie 1986].

Promiscuous AR
AR promiscuity, or altered specificity of AR binding, is an additional
mechanism through which prostate cancer cell signaling and growth can occur under
castrate levels of androgen. This modified specificity may be due to gene mutations,
such as T877A or L701H, which allow binding of non-androgenic ligands, giving
11
rise to stimulation of AR activity by molecules which are not expected to have an
agonistic effect on the AR in signaling pathways, such as non-androgen steroids and
androgen antagonists [Gaddipati 1994, Suzuki 1993]. It has recently been shown
that prostate cancer cells expressing the T877A mutation exhibit increased cell
proliferation and elevated androgen-independent AR transcriptional activity than
cells with wild-type AR, in hormone depleted conditions [Sun 2006]. Tilley et al
reported that presence of mutations in the AR gene is associated with failure of
hormonal therapy [Tilley 1996]. Further, it has been shown that several
antiandrogens, including hydroxyflutamide, can induce AR-associated transcription
and stimulate prostate cancer cell proliferation [Veldscholte 1992]. It has also been
suggested that aberrant expression of AR coactivators may contribute to a
promiscuous AR, where overexpression of coactivators increases transactivation of
the AR at physiological concentrations of adrenal androgen [Gregory 2001].

Outlaw Mechanisms
The outlaw pathway functions independently of androgen, but fundamentally
relies on downstream AR activation. Outlaw mechanisms are characterized by
ligand-independent AR activation, by either non-androgenic growth factors or
tyrosine kinase receptors via alternate growth pathways such as the Akt and/or
MAPK pathways [Sherwood 1998, Nickerson 2001, Culig 1994, Craft 1999, Yeh
1999, Wen 2000]. Culig et al reported the activation of AR via signaling by insulin-
like growth factor-1 (IGF-1), keratinocyte growth factor (KGF), epidermal growth
12
factor (EGF), and interleukin-6 (IL-6), where the AR antagonist bicalutamide blocks
growth factor-stimulated activation of AR, suggesting the intrinsic role of the AR in
the outlaw pathway [Culig 1994].
Receptors of the EGFR family (EGFR, Her-2/neu, HER3, HER4) belong to
the superfamily of receptor tyrosine kinases (RTK) and have regulatory roles in cell
growth and differentiation [Zwick 2001]. The binding of natural ligands, such as
EGF, TGF-α, and heregulin, which provide stimulatory signals for dimerization and
autophosphorylation of these RTKs, results in downstream cytoplasmic signaling,
activation of intracellular signal transduction cascades, and regulation of target gene
transcription [Klapper 2000, Olayioye 2000].
The HER-2/neu (c-erb B-2) proto-oncogene located on chromosome 17q
encodes the 185 kDa RTK, Her-2/neu, which is associated with multiple signal
transduction pathways [Ross 1998]. The associations between Her-2/neu and AR-
activating pathways such as MAP kinase have been reported, suggesting mechanisms
through which Her-2/neu can activate the androgen receptor in the absence of
androgen and enhance the magnitude of response of the AR, even at low levels of
androgen [Craft 1999, Yeh 1999]. Berger et al have recently shown that androgen
depletion results in significantly elevated Her-2/neu expression in tumors in vivo,
which is accompanied by a corresponding increase in serum PSA and is inversely
associated with serum testosterone [Berger 2006]. Her-2/neu and Her3 have also
been shown to contribute to growth in recurrent prostate cancer cells [Gregory 2005].
In addition, several reports have identified the association of Her-2/neu
13
overexpression with initial exposure to androgen ablation therapy and the
development of CRPC [Shi 2001, Signoretti 2000, Osman 2001].
AR activation may occur through activation of alternate growth pathways
such as the MAP kinase or PI3K/Akt pathways. Activated MAP kinase is
upregulated in prostate cancer cells in response to androgen deprivation [Lee 2003]
and Her2/neu-induced transactivation of the AR has been shown to be mediated
through the MAP kinase pathway [Yeh 1999]. Activation of the PI3K/Akt pathway
regulates various intracellular biological processes, such as proliferation and
survival, through phosphorylation of downstream targets [Coffer 1998, Datta 1999].
Inactivation of the tumor suppressor phosphatase and tensin homolog (PTEN)
relieves its inhibitory effects on the PI3K/Akt pathway and may be an additional
mechanism through which prostate cancer cells avoid apoptosis in CRPC [Li 1997].

AR Independent Signaling
Some cancer cells inherently possess the ability to evade cell death and
survive even severe conditions of cellular stress. A clinical manifestation of this is
the recurrence of prostate cancer following androgen deprivation therapy. Thus, key
molecules involved in the inhibition of apoptosis may likely contribute to prostate
cancer cell survival upon androgen withdrawal and the subsequent development of
castration resistant growth. In contrast to AR-activation dependent mechanisms,
upregulation of signals which are intrinsically anti-apoptotic may contribute to
14
CRPC development in a manner which bypasses pathways leading to downstream
AR activity.
The potential mechanisms of AR bypass, such as upregulation of stress
response and antiapoptotic signaling, may not be mutually exclusive from AR
dependent pathways leading to androgen independent growth. Rather, it is plausible
that they may work together to contribute to the development of androgen
insensitivity. Cai et al found that Grp78, functioning as an ER chaperone, forms a
complex with EGFR in human epidermoid carcinoma cells maintained in glucose-
starved conditions, and suggests this direct interaction as a mechanism of protection
in cancer cells under glucose starvation [Cai 1998]. In addition, Shi et al showed
that forced expression of Her-2/neu was directly accompanied by the upregulation of
Bcl-2 [Shi 2006]. Thus, signals traditionally thought to be involved with or
independent of androgen receptor signaling may actually interact to regulate cell
activity.
Molecular signaling differs in cells which require greater than castrate levels
of androgen to survive and those which do not. These changes may result as a
modification of intracellular machinery allowing survival upon androgen withdrawal,
or cells may selectively survive following androgen deprivation due to existing
alterations. Regardless of the implications of causal induction or clonal selection,
molecular changes contributing to the development of castration resistance can also
be viewed as either contributing to the AR dependent pathway or independent of AR
activation. Interestingly, these pathways may not be mutually exclusive.
15
1.3 Stress Response Proteins
1.3.1 Overview of Stress Response Protein Family
Mammalian cells utilize mechanisms that allow the ability to withstand
adverse environmental conditions. This may manifest in biochemical or molecular
changes such as upregulation of proteins that provide protection to the cells. Two
major families of proteins that contribute to these protective functions are the heat
shock proteins (HSPs) and the glucose regulated proteins (GRPs).
A number of stress-induced proteins have been implicated in the
development of CRPC. Heat shock protein Hsp70 shares 60% homology with stress
response protein glucose-regulated protein 78 (Grp78), and has been shown to be
highly expressed in castration resistant prostate cancer cells, where Hsp70
suppression in these cells results in downregulation of Bcl-2, inhibition of cell
growth, and induction of apoptosis [Munro 1986, Zhao 2004]. Inhibition of heat
shock protein 27 (Hsp27), another molecular chaperone, has also been shown to
increase apoptosis and decrease cell growth in castration resistant prostate cancer
cells [Rocchi 2004]. Hsp27 was further found to be an independent predictor of
clinical outcome in prostate cancer, where increased Hsp27 expression is highly
associated with reduced overall survival [Cornford 2000]. In addition, heat shock
protein 90 (Hsp90) interacts with several signaling proteins associated with CRPC,
including Her-2/neu, Akt, and androgen receptor (AR) [Neckers 2002, Xu 2001, Sato
2000, Marivoet 1992]. Solit et al demonstrated that treatment of prostate cancer
xenograft tumors with 17-allylamino-17-demethoxygeldanamycin (17-AAG), a
16
small molecule inhibitor of Hsp90, results in suppression of growth in both androgen
dependent and castration resistant prostate cancers, with corresponding reductions in
Her-2/neu, Akt, and AR [Solit 2002]. Thus, stress-response proteins appear to
contribute substantially to prostate cancer progression and the development of
CRPC, where suppression of these proteins is inhibitory to castration resistant
growth. The application of these signals as potential therapeutic targets may allow
cells to become susceptible to environmental stresses.

1.3.2 The Unfolded Protein Response
The endoplasmic reticulum (ER) is responsible for post-translational
modifications, folding, and oligomerization of newly synthesized proteins which are
translocated into the ER lumen. There remains constant intracellular communication
between the ER, cytoplasm, and nucleus to maintain homeostatic balance in response
to the various external stimuli cells may experience. Response to environmental
changes may result in alterations in gene expression, thus promoting adaptation to
these stimuli or progression to apoptosis. Proteins termed molecular chaperones,
located in the ER lumen, play important roles in proper protein folding and
preservation of ER homeostasis. One of the best studied chaperone families which
reside in the ER are the glucose regulated proteins (GRPs), which were named as
such due to their induced expression upon glucose starvation [Lee 1987]. The GRPs
are constitutively expressed in all cells, and various stimuli may result in induction
of these proteins, including calcium depletion from the ER lumen, improper
17
expression of proteins or protein subunits, and unfolded and misfolded protein
accumulation [Kaufman 1999]. Upon external stimulation, a signal is sent to
activate transcription of GRPs and other related genes, and this is termed the
unfolded protein response (UPR). The UPR is a process conserved from yeast to
humans which acts to maintain homeostasis of the ER. The UPR involves
attenuation of new protein synthesis, degradation of misfolded proteins, and
signaling for the initiation of apoptosis [Kaufman 1999]. Stimuli that have been
shown to activate the UPR include tunicamycin, heavy chain immunoglobulin
expression, brefeldin A, Ca
2+
ionophores, and heavy metal ions [Kaufman 1999].
Several different genes may be induced through the UPR, including GRP78, GRP94,
calreticulin, CHOP/GADD153, FKBP13, and HSP47 [Kaufman 1999].

1.3.3 Grp78
A potential cellular survival mechanism contributing to the bypass of AR in
CRPC is through upregulation of stress response pathways, which confer protection
to cells when subjected to adverse conditions. The stress-responsive glucose-
regulated proteins (GRPs) were initially identified when cells underwent glucose
deprivation [Shiu 1977, Lee 1987]. The best-examined member of the GRP family is
Grp78, a 78-kDa protein also recognized as immunoglobulin heavy-chain binding
protein (BiP) [Haas 1983]. Normal functions of Grp78, which is generally localized
to the endoplasmic reticulum (ER), include proper folding and assembly of
polypeptides leading to formation of functional proteins, and retention of
18
unassembled precursors or proteins to the ER [Little 1994, Dorner 1988].
Upregulation of Grp78 is a component of the unfolded protein response (UPR) of
cells exposed to conditions of stress [Lee 2001, Kaufman 1999].
The involvement of Grp78 in enhanced cell survival is suggested by the
remarkable elevation of Grp78 transcription rates under various stress conditions
[Lee 1987]. Recently, Grp78 has been shown to directly interact with intermediates
of the apoptotic pathway (caspases 7 and 12) by blocking caspase activation, where
Grp78 induction results in increased cell survival and inhibition of apoptosis
[Miyake 2000, Reddy 2003, Rao 2002]. As an ER chaperone, Grp78 is also an
essential component of the unfolded protein response of cell survival mechanisms
[Lee 2001]. The anti-apoptotic functions of Grp78 indicate its potential role in cancer
progression and resistance to therapies, and the association between increased Grp78
and malignancy has previously been implicated in various cancer cell lines and
tumors [Mintz 2003, Lee TIBS 2001, Patierno 1987, Bini 1997, Gazit 1999].
Miyake et al reported that Grp78 prevents stress-induced apoptosis in
prostate cancer cells, and that induction of Grp78 is diminished in the presence of
androgen [Miyake 2000]. Thus, in a stress environment characterized by androgen
ablation, it is intuitive that Grp78 expression would be induced as a mechanism of
cell survival in the development of CRPC. Recently, Grp78 reactivity has been
identified as a putative marker of CRPC [Mintz 2003]. Clinical studies examining
Grp78 expression in human prostate cancer samples have demonstrated that
increased levels of Grp78 are significantly associated with castration resistant status
19
[Pootrakul 2006]. This clinical observation was confirmed in an in vitro cell line
model of cells grown in androgen-rich and androgen-deprived conditions, which
mimics the clinical transition of localized prostate cancer to CRPC [Pootrakul 2006,
Shi 2006].

1.4 Apoptosis
1.4.1 Overview
Apoptosis is the process of programmed cell death by which tissues maintain
homeostasis by balancing cell proliferation and cell death. There are specific
biochemical and morphological changes which characterize apoptosis, including cell
shrinkage, nuclear blebbing, and DNA fragmentation. Major mediators of apoptosis
are the caspases, which are a family of intracellular cysteine proteases with
substrates contributing to DNA repair, cytoskeletal maintenance, nuclear integrity,
and cell survival [Goyal 2001]. Caspases 1, 2, 8, 9, and 10 are thought to be
initiators of apoptosis, and caspases 3, 6, and 7 function in the execution of apoptosis
[Datta 1999]. There are two main pathways through which apoptosis proceeds
(Figure 1-2). The extrinsic pathway is initiated by death receptors (TNF-α receptor,
CD95) located on the cell surface, and the intrinsic pathway is stimulated by
alterations to normal mitochondrial function [Krammer 2000, Wang 2001].
There are two major gene families involved in the regulation of apoptosis,
including the BCL2 family and the inhibitors of apoptosis (IAP). Members of the
BCL2 family may demonstrate pro- (BAX, BAD, BAK) or anti- (BCL2, BCL-X
L
)
20
apoptotic function [Altieri 2003]. BCL2 signals are able to homodimerize or
heterodimerize at the mitochondrial membrane and affect mitochondrial
permeability, which, if increased, will facilitate caspase activation and allow
apoptosis to occur [Cory 2002].

1.4.2 Inhibitors of Apoptosis
The IAPs were originally identified in baculovirus as inhibiting host viral
response to viral infection, and have now been shown to block cell death in
mammalian cells by inhibiting the action of caspases [Deveraux 1997]. The
members of the inhibitor of apoptosis (IAP) gene family share structural similarities,
the most significant of which is the Baculovirus IAP Repeat (BIR), 1-3 copies of a
70 amino acid zinc finger fold [Altieri 2003]. Some members of the IAP family also
contain a caspase-recruitment domain (CARD), a RING finger, a ubuiquitin-
conjugating domain, and a nucleotide-binding P loop motif [Deveraux 1999].
Caspases are intracellular cyteine proteases that become active following proteolysis
and cleave cellular proteins involved in cytoskeletal integrity, cellular repair, and
energy production, thereby contributing to the executioner phase of apoptosis [Altieri
2003]. Eight members of the IAP family have been identified in humans, of which
the best examined are XIAP, c-IAP1, c-IAP2, and Survivin [Altieri 2003]. IAPs are
able to counteract pro-apoptotic actions by inhibiting caspases, but may also be
inhibited by protein released from the mitochondria, such as SMAC/DIABLO, which
cause their release from caspases [Shi 2002].
21
Figure 1-2. Major pathways in apoptosis
The extrinsic, or death-receptor pathway, acts through caspase 8 to initiate apoptosis.
The intrinsic, or mitochondrial pathway, is mediated by caspase 9, but both pathways
will meet when effector caspases are activated. Apoptosis is also regulated by the
BCL2 family, which can modify mitochondrial membrane permeability by halting or
promoting the release of cytochrome c. Additional regulators of programmed cell
death include the inhibitors of apoptosis (IAPs), which act downstream of BCL2
signals to prevent caspase activity [Altieri 2003].

*Adapted from Altieri 2003

Survivin
22
1.4.3 Survivin
Survivin, a 16.5-kDa protein, is the smallest member of the ‘inhibitor of
apoptosis’ (IAP) family [Ambrosini 1997] and has been identified as a nuclear as
well as cytosolic protein [Fortugno 2002]. While Survivin is highly expressed in
embryonic development, it is generally undetectable in terminally differentiated
normal tissue [Ambrosini 1997, Adida 1998, Kobayashi 1999]. Distinct functions
have been identified for Survivin, including mitotic regulation and cell death
inhibition. Survivin expression and activity is crucial for proper mitotic progression
[Uren 2000, Kallio 2001]. Further, mechanistic evidence of an anti-apoptotic role
has been reported by Shin et al, who describe the direct inhibition of caspases 3 and
7 by Survivin [Shin 2001].
The intrinsic functions of Survivin demonstrate a prospective role in cancer
cell survival. In the development of CRPC, Survivin would likely contribute via the
AR bypass pathway. Overexpression of Survivin has previously been reported in a
number of hormonally influenced cancers, including cancers of the breast, uterus,
and ovaries [Tanaka 2000, Saitoh 1999, Yoshida 2001]. Further, cytoplasmic
expression of Survivin has previously been associated with poorer prognosis in a
number of malignancies, including neuroblastoma, colorectal, and bladder cancers
[Adida 1998, Kawasaki 1998, Swana 1999]. It has recently been demonstrated that
Survivin promotes resistance to antiandrogen treatment in prostate cancer, both in
vitro and in vivo [Zhang 2005]. Increased Survivin expression is seen in clinical
prostate cancer samples, as compared to control tissue [Kishi 2004, Kaur 2004,
23
Shariat 2004]. Fromont et al have also shown significantly elevated Survivin gene
expression in CRPC compared to localized prostate cancer [Fromont 2005], in a
study which included a cohort of 33 stage T
2
and T
3
patients, and 13 CRPC patients.

1.5 The Her-2/Neu Pathway
Receptors of the EGFR family (EGFR, Her-2/neu, HER3, HER4) belong to
the superfamily of receptor tyrosine kinases (RTK) and have regulatory roles in cell
growth and differentiation [Zwick 2001]. The binding of natural ligands, such as
EGF, TGF-α, and heregulin, which provide stimulatory signals for dimerization and
autophosphorylation of these RTKs, results in downstream cytoplasmic signaling,
activation of intracellular signal transduction cascades, and regulation of target gene
transcription [Klapper 2000, Olayioye 2000].
The HER-2/neu (c-erb B-2) proto-oncogene located on chromosome 17q
encodes the 185 kDa RTK, Her-2/neu, which is associated with multiple signal
transduction pathways [Ross 1998]. The associations between Her-2/neu and AR-
activating pathways such as MAP kinase have been reported, suggesting mechanisms
through which Her-2/neu can activate the androgen receptor in the absence of
androgen and enhance the magnitude of response of the AR, even at low levels of
androgen [Craft 1999, Yeh 1999]. Berger et al have recently shown that androgen
depletion results in significantly elevated Her-2/neu expression in tumors in vivo,
which is accompanied by a corresponding increase in serum PSA and is inversely
associated with serum testosterone [Berger 2006]. Her-2/neu and Her3 have also
24
been shown to contribute to growth in recurrent prostate cancer cells [Gregory 2005].
In addition, several reports have identified the association of Her-2/neu
overexpression with initial exposure to androgen ablation therapy and the
development of CRPC [Shi 2001, Signoretti 2000, Osman 2001]. Further evidence
of Her-2/neu involvement in prostate cancer cell growth has been demonstrated by
upregulation of growth pathway signaling downstream of Her-2/neu. Activated
MAP kinase is upregulated in prostate cancer cells in response to androgen
deprivation [Lee 2003] and Her2/neu-induced transactivation of the AR has been
shown to be mediated through the MAP kinase pathway [Yeh 1999].

1.6 Cell Cycle
1.6.1 Overview
It has been well established that tumors arise from deregulated cell growth.
Normal cell growth and proliferation is strictly regulated by the cell cycle, which
includes two major phases, interphase and mitosis, repeated in sequence. Interphase
includes G1 phase (gap preceding DNA replication, where cells is preparing for
DNA synthesis), S phase (doubling of DNA), and G2 phase (gap following DNA
replication involving cell growth and preparation for cell division). Mitosis includes
the M phase (where the process of nuclear cell division occurs). Before committing
to DNA replication, cells in G1 can enter the G0 resting state, and these cells are
neither growing nor proliferating [Vermeulen 2003].
25
Tight control of the cell cycle progression is dependent on a family of
proteins call the cyclin-dependent kinases (CDKs), a family of serine/threonine
protein kinases that are activated throughout the cell cycle. CDKs which are active
during the cell cycle include CDK1, CDK2, CDK4, and CDK6. There are three
cyclin D proteins (D1, D2, and D3) that are essential for regulation of the G1 phase,
and form CDK-cyclin D complexes with CDK2 and CDK4 [Sherr 1994]. In
addition to interactions with cyclins, regulation of the CDKs is also maintained by
the cyclin-dependent kinase inhibitors (CKIs) which can bind to the CDK alone or to
CDK-cyclin complexes. These include the Cip/Kip family (p21, p27, p57), which
inhibit G1 CDK-cyclin complexes, and the INK4 family (p15, p16, p18, p19), which
inactivate G1 CDK and are responsible for inactivation by direct binding [Grana
1995].
The implications of loss of cell cycle regulation in tumor growth have lead to
these signals being the focus of numerous studies in cancer development and
progression, in particular, those markers involved in the advancement from G1 to S
phase. One of the best studied regulators of G1/S transition is p53. The role of p53
in cancer is founded by its integral regulation of the cell cycle, apoptosis, DNA
repair, and response to DNA damage. It has been demonstrated that p53 gene
mutations result in nuclear accumulation of the altered p53 protein [Esrig 1993]. In
addition, many tumors carry a mutated p53 gene, and the majority of human cancers
have malfunctioning p53 protein [Vogelstein 2000]. Studies in prostate cancer have
also demonstrated potential involvement in the development of castration resistance.
26
Burchardt et al have shown that downregulation of p53 function in LNCaP cells,
either by expression of antisense p53 cDNA or dominant-negative p53, results in the
formation of tumors following inoculation of the cultured cells into castrated mice,
where incoculation with parental LNCaP cells does not [Burchardt 2001]. Studies in
which immunohistochemical analyses were performed on samples of prostate tumors
from men with androgen dependent and castration resistant prostate cancer
demonstrated that while nuclear accumulation of p53 was a rare event in androgen
dependent prostate cancer, nearly 50% of castration resistant tumors exhibited p53
nuclear accumulation [Salem 1997, Navone 1993].
An additional G1/S regulator demonstrating particular significance in tumor
growth is p27. Cell cycle regulator p27 is a member of the kip/cip family of cyclin-
dependent kinase inhibitors and aids in the control of the cell cycle G1/S transition
by inhibiting the kinase activity of cyclin E/CDK2 complex necessary for
retinoblastoma (Rb) protein phosphorylation [Cordon-Cardo 1995]. By maintaining
the dephosphorylated status of Rb, p27 halts cell cycle progression at the G1/S
transition. It has been reported that loss of p27 expression is associated with the
progression of several types of tumors, including breast, colon, and prostate
[Catzavelos 1997, Loda 1997, Porter 1997, Cote 1998], suggesting that the loss of
p27 may contribute to cancer progression via uncontrolled cell proliferation. There
are clinical and in vitro data demonstrating that p27 expression is initially increased
in prostate cancer cells upon androgen deprivation, and that the transition from
androgen dependent growth to castration resistant growth is accompanied by
27
decreased p27 expression [Shi 2006]. Thus, the initial antiproliferative response to
androgen ablation therapy appears to be due in part to an increase in p27 expression,
which may result in cell cycle arrest in androgen-responsive prostate cancer cells. It
is clear that examining the involvement of cell cycle regulators in prostate cancer cell
escape from androgen dependence is particularly useful and may suggest important
and rational therapeutic targets.

1.6.2 Cyclin D1
The cyclins are a family of cell cycle regulatory proteins that modulate the
activity of the family of serine/threonine kinases, termed CDKs [Pestell 1999].
Cyclin D1 functions as the rate-limiting regulatory subunit of a multi-protein
cyclin/CDK complex that is critical for controlling progression through the G1- to
S- phase of the cell cycle [Pestell 1999]. Cyclin D1 functions by binding to either
cyclin dependent kinase 4 (CDK4) or CDK6, thereby activating the unit [Knudsen
2006]. Activated complexes can subsequently phosphorylate other signals that are
critical to regulating the progression from G1 to S.
A well-established substrate of the cyclin D1-CDK4/6 complexes is the
retinoblastoma tumor suppressor protein (RB) [Kato 1993]. When RB is
hypophosphorylated, it forms complexes which repress transcription of genes
required for G1-S transition [Sherr 2002]. Once phosphorylated by the cyclin D1-
CDK complexes, the transcriptional repressor function of RB is removed and the
cell cycle progresses [Knudsen 2006]. Activation by the cyclin D1-CDK
28
complexes have been shown to be critical to this process, as demonstrated in
studies where tumor cells lacking RB expression have decreased dependency on
cyclin D1 [Muller 1994, Lukas 1995], and in the findings that cyclin D1-CDK4
inhibitor, p16ink4a, is ineffective in cells deficient in RB [Lukas 1995]. These
studies suggest that a major mechanism of cell cycle regulation is through the
cyclin D1 activation of CDK4/6 kinase activity [Knudsen 2006]. Cyclin D1 may
also exert control over the cell cycle via CDK kinase-independent functions. CDK
inhibitors p21 and p27 may be sequestered by cyclin D1, promoting G1-S
progression [Sherr 1999].
Deregulation of cyclin D1 gene expression has been described in numerous
types of human tumors and the tissue specific dependency of certain oncogenes on
cyclin D1, including Ras, ErbB-2 and b-catenin [Lee 2000, Yu 2001, Hulit 2004] is
well documented.
Cyclin D1 mRNA levels were induced by EGF in human prostate cancer cell
lines [Perry 1998] and were found to be increased in both primary prostate cancer
samples [Han 1998] and androgen-independent bone metastases [Drobnjak 2000].
Increased expression of cyclin D1 in LNCaP human prostate cancer cells enhanced
cell growth and tumorigenicity [Perry 1998, Chen 1998] while inhibition of ErbB-
2/ErbB-3 signaling by the flavonoid, quercetin, reduced cyclin D1 protein levels,
resulting in an inhibition of cell cycle progression both in prostate cancer cell lines
[Huynh 2003] and in vivo [Ma 2004]. These data are all consistent with a role for
29
cyclin D1 as a mediator of prostate epithelial cell proliferation and prostate cancer
cell growth.

1.7 Recent Novel Findings in Prostate Cancer
It has been widely accepted that chromosomal translocations contribute to the
development of hematologic malignancies and are rarely associated with epithelial-
derived tumors. However, in 2005, Tomlins et al reported findings that TMPRSS2
fusions are commonly found in prostate cancer. Using their developed method
termed cancer outlier profile analysis (COPA) to identify outlier gene profiles of
recurrent rearrangements or remarkable amplification for specific cancers, Tomlins
et al identified recurrent gene fusions of the 5’ untranslated region of the TMPRSS2
gene to two ETS transcription factors, ERG and ETV1 in prostate cancer tissues
[Tomlins 2005]. It was subsequently shown, using xenograft models of androgen-
dependent and castration resistant prostate cancer, that the TMPRSS2:ERG fusion is
present in both androgen-dependent and androgen receptor-negative castration
resistant tumors, although the fusion gene is not expressed in androgen receptor-
negative castration resistant specimens [Hermans 2006]. Jia et al also demonstrated
that TMPRSS2, unlike other AR target genes, did not have similar levels of
chromatin remodeling and increased gene expression in castration resistant prostate
cancer cells in the absence of ligand [Jia 2006].

30
1.8 Purpose of Study
Growth and proliferation of prostate cancer cells are regulated by the
presence of androgen. Thus, androgen ablation therapy is considered the mainstay of
treatment for prostate cancer. Androgen dependent tumors are characterized by the
ability of tumor cells to undergo cell death upon androgen withdrawal. Some cancer
cells, however, maintain the ability to survive even in an androgen depleted
environment, and these cells subsequently proliferate into castration resistant tumors.
The exact mechanisms causing the transition from androgen dependence to
castration resistance are unclear, and are thus the focus of these and other studies.
Because the distinguishing factor between androgen dependent prostate
cancer cells and castration resistant cells is the ability to survive upon stress
conditions such as androgen deprivation, there is strong interest in signaling involved
in cell survival and cellular response to stress environments. Studies in our lab and
others have suggested that two such proteins, Grp78 and Survivin, are overexpressed
in castration resistant patients as compared to those who are responsive to androgen
ablation therapy, and that downregulation of these signals leads to markedly
increased apoptosis in cancer cells [Pootrakul 2006, Mintz 2003, Fromont 2005,
Dong 2005, Zhang 2005]. To further investigate the significance of these findings,
we developed a unique clinical cohort designed to correspond to the successive
stages in the development of CRPC, including tumors from patients not exposed to
anti-androgen therapy (untreated), patients who were exposed to anti-androgen
therapy preoperatively (treated), and patients with CRPC. We have also applied the
31
use of an in vitro system which mimics the clinical model and allows direct
evaluation of molecular changes that occur under various cell growth conditions.
Our hypothesis is that signals promoting anti-apoptotic processes, such as
stress response protein Grp78 and Survivin, are crucial for the development of
castration resistance in prostate cancer, where cancer cells are able to survive despite
harsh environmental condition such as androgen withdrawal. We further propose
that inhibition of these signals will alter the ability of prostate cancer cells to
maintain castration resistant growth, and that combined targeting of anti-apoptotic
signals along with stimulators of growth and proliferation (tyrosine kinase receptor
Her-2/neu) shown to be important in castration resistance will result in synergistic
effects on tumor cell death. These studies are being performed for the purpose of
exploring which molecular mechanisms are critical to prostate cancer progression
and the development of CRPC, using a unique clinical model designed to represent
the development of CRPC and an in vitro system to investigate the meaning of our
clinical observations. Our studies will allow us to have a better understanding of the
progression to CRPC, and to evaluate if survival signals may serve as rational
therapeutic targets for the management of castration resistance.

32
Chapter Two: Increased Grp78 Expression is Associated with Prostate Cancer
Progression and the Development of Castration Resistance

2.1 Introduction
Resistance to castration therapies persists as the predominant challenge in the
treatment of advanced prostate cancer. Androgen dependent prostate cancer is
characterized by the ability of cancer cells to undergo apoptosis in response to
hormone depletion. The transition to castration resistant prostate cancer (CRPC)
requires the survival of tumor cells in such conditions, which may be attributed to a
number of molecular mechanisms resulting in the evasion of apoptosis. One
potential cellular survival mechanism in CRPC is through upregulation of stress
response pathways, which confers protection to cells when they are subject to
adverse conditions.
The glucose-regulated proteins (GRPs) were initially identified as such in
transformed chick embryo fibroblasts growing in glucose-deprived medium [Shiu
1977, Lee 1987]. The best examined member of the GRP family is Grp78, a 78-kDa
protein also recognized as immunoglobulin heavy-chain binding protein (BiP) [Haas
1983]. Normal functions of Grp78, which resides in the endoplasmic reticulum (ER)
lumen, include proper folding and assembly of other polypeptides leading to the
formation of functional proteins, retention of unassembled precursors to the ER,
targeting misfolded proteins for degradation, ER Ca
2+
binding, and the regulation of
trans-membrane ER stress inducers [Little 1994, Dorner 1988, Lee 2005].
33
The involvement of Grp78 in enhanced cell survival is suggested by the
remarkable elevation of GRP78 transcription rates under various environmental
stress conditions [Lee 1987]. Recently, Grp78 has been shown to directly interact
with intermediates of the apoptotic pathway, blocking caspase activation, where
Grp78 induction results in increased cell survival and inhibition of apoptosis [Reddy
2003, Rao 2002, Miyake 2000]. As an ER chaperone, Grp78 is a key component of
the unfolded protein response, promoting cell survival under ER stress [Lee 2001,
Kaufman 1999]. The inherent roles and anti-apoptotic capabilities of Grp78 indicate
a potential role in cancer progression. Suppression of Grp78 level by antisense in
fibrosarcoma results in inhibition of tumor growth [Jamora 1996]. Elevation of
Grp78 in the microenvironment of tumors due to nutrient deprivation or hypoxia
confers survival advantage to cancer cells and leads to resistance to therapeutics
[Dong 2005]. The association between increased Grp78 and malignancy has
previously been implicated in various cancer cell lines and tumors [Lee 2001, Mintz
2003, Bini 1997, Gazit 1999, Fernandez 2000]. Grp78 serum reactivity has recently
been identified in patient sera as a putative marker of CRPC [Mintz 2003]. We were
interested in assessing the prospective role of Grp78 in prostate cancer progression
and the development of CRPC.

34
2.2 Materials and Methods
2.2.1 Patient Population
The recruitment and studies of patients described here have been approved by
local institutional review boards. This study included tumor samples from 219
patients with prostate cancer, comprised of 3 distinct cohorts of patients. 191
patients were classified as pathological stage T
3
N
0
M
0
disease [UICC 1992] and
specimens were obtained through radical retropubic prostatectomy with bilateral
pelvic lymph node dissection at the University of Southern California/Norris
Comprehensive Cancer Center between 1982 and 1996. These patients were further
subdivided according to treatment status. Treatment consisted of neoadjuvant
androgen ablation therapy with 1 mg diethylstilbestrol 2 or 4 times per day for 3 days
to 20 weeks before radical prostatectomy. The stage T
3
N
0
M
0
untreated group
included 164 patients who were not exposed to pre-operative androgen ablation
therapy. The group of 27 men comprising the stage T
3
N
0
M
0
treated group had
received pre-operative neoadjuvant androgen ablation therapy. All stage T
3
N
0
M
0

patients were considered responsive to anti-androgen therapy [Fradet 1996]. Tumor
samples were obtained from 28 patients with CRPC who underwent hormone
ablation via orchiectomy and systemic hormone therapy but continued to
demonstrate rising prostate specific antigen (PSA). Between 1990 and 1992, these
men underwent transurethral resection to relieve urinary obstruction at Ruhr
University, Bochum, Germany. All tumor grading was in concordance with the
Gleason system [Gleason 1977].
35
2.2.2 Patient Followup
Evaluations of all patients were done at 1, 2, and 6 months postoperatively, at
6-month intervals for 5 years following surgery, then yearly. Biopsy was used to
assess clinical recurrence of prostate cancer, and metastatic disease was determined
according to bone scan or alternate clinical findings. PSA recurrence was designated
to patients demonstrating serum PSA levels of ≥0.4ng/mL on 2 consecutive tests.
Median follow-up in the untreated group of 164 patients was 12.7 years with a range
of 1.6 to 20 years. Median age was 67 years, ranging from 47 to 81 years.

2.2.3 Immunohistochemistry (IHC)
Formalin fixed 5 micrometer sections were taken from paraffin-embedded
prostate cancer specimens and cell lines and mounted on poly-L-lysine coated slides.
The slides were deparaffinized in xylene, washed with 100% ethanol, followed by
rehydration in 95% ethanol. 3% hydrogen peroxide in absolute methanol was used
to quench endogenous peroxidase. Antigen retrieval was performed using citrate
buffer (pH=6) and microwaving for 30 minutes [Shi 1997], followed by cooling at
room temperature for 20 minutes. The slides were then blocked with normal horse
serum for 20 minutes, and incubated for 1 hour with anti-Grp78 rabbit polyclonal
antibody (Santa Cruz Biotech, Santa Cruz, CA) at a 1:100 dilution in phosphate-
buffered saline. Incubation with biotinylated horse anti-rabbit secondary antibody at
a 1:200 dilution was followed by avidin-biotin-conjugate (ABC, Vector
Laboratories, Inc., Burlingame, CA). Chromogen of 0.03% diaminobenzidine was
36
then applied, with hematoxylin counterstaining. Negative controls consisting of
diluent with no antibody, and positive prostate cancer controls with heterogeneous
immunoreactivity were used in all experiments.
Cultured prostate cancer cells (LNCaP, C42B) were harvested, cytospun on
poly-L-lysine coated slides at 250,000 cells per slide, and formalin-fixed. Antigen
retrieval was performed using citrate buffer (pH=6) and microwaving for 5 minutes
[Shi 1997], followed by cooling at room temperature for 15 minutes. Subsequent
steps in IHC protocol follow as described above.

2.2.4 Immunoreactivity Assessment of Clinical Samples
All slides were interpreted by two pathologists (SRS, DH), who were blinded
to all outcome data. Tumor scores were categorized based on 2 criteria: 1) percent of
tumor cells demonstrating cytoplasmic immunoreactivity and 2) intensity of
cytoplasmic immunostaining. For assessment according to percent of cytoplasmic
reactivity, tumors were classified as showing low Grp78 expression (<50%), or high
Grp78 expression (>50%). For intensity of cytoplasmic immunoreactivity, tumors
were classified as having low Grp78 expression (1+), moderate Grp78 expression
(2+), or high Grp78 expression (3+). Grp78 status was assigned as negative to cases
with <10% Grp78 immunoreactivity or weak (1+) staining. All other cases were
assigned positive Grp78 status. Upon identification of focal areas where Grp78
expression levels were markedly intense, tumors were further categorized by percent
of cells demonstrating intense (3+) Grp78 immunoreactivity (<5%, low Grp78; >5%,
37
high Grp78). Due to the heterogeneity of Grp78 immunoreactivity, scoring
corresponds with an overall evaluation of the entire tissue section. Lymphocytes,
which are highly immunoreactive with anti-Grp78, were used as internal positive
controls.

2.2.5 Cell Culture
LNCaP and C42B cells were grown in RPMI 1640 medium (Invitrogen,
Carlsbad, CA) with 50units/mL penicillin, 50 units/mL streptomycin, and 10% fetal
calf serum (FCS) (Mediatech, Inc. Herndon, VA). For preparation of androgen-
depleted medium, FCS and RPMI 1640 were replaced by 10% dextran/charcoal-
stripped serum (CSS) (Omega Scientific Inc, CA) and phenol-free RPMI 1640
(Invitrogen, Carlsbad, CA), as previously described in the literature [Craft 1999].
All cell lines were maintained in a humidified incubator at 5%CO
2
and 37°C.

2.2.6 Automated Cellular Imaging
Immunostaining and evaluation of immunostained cell lines were carried out
in triplicate, where immunoreactivity was assessed using ACIS II (Clarient, Inc.,
Aliso Viejo, CA) [Bauer 2000]. The ACIS II system consists of a computer-assisted
bright-field microscope (4X, 10X, 20X, 40X, 60X objectives) coupled to a SONY 3-
chip CCD camera. This fully automated system creates a reconstructed image of an
IHC stained slide, and uses wavelength specific technology to detect color
differences between objects. Immunostained slides of cytospun cell lines were
38
scanned at 4X magnification, followed by image capture, transformation to pixels,
and quantification by hue (color), saturation (color purity), and luminosity
(brightness). Five regions of interest at 4X magnification were manually selected for
each sample slide and brown color (DAB chromogen) was assessed by ACIS
software which counts pixels based on 256 levels of color intensity. Representative
areas were analyzed for intensity and percent of cells positive for brown color.

2.2.7 Western Blot Analysis
For western blot analysis, cell lysates from LNCaP and C42B cells were
prepared by lysing in 1mL ice-cold RIPA buffer. Equal amounts of total protein
from each sample were subjected to SDS-PAGE in a 7.5% Tris-HCl gel (Bio-Rad
Laboratories, Hercules, CA). Following electrophoresis, the proteins were
transferred to a pure nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA).
The membrane was then incubated in Odyssey Blocking Buffer (Li-Cor Biosciences,
Lincoln, Nebraska), followed by overnight incubation with primary rabbit polyclonal
anti-Grp78 antibody (1:500 dilution; Santa Cruz Biotechnology Inc, Santa Cruz,
CA). Signal detection was performed using Alexa Fluor 680 goat anti-rabbit
antibody (Molecular Probes, Eugene, Oregon) and subsequent scanning of the
membrane by the Odyssey Infrared Imager (Model 9120, Li-Cor Biosciences,
Lincoln, Nebraska). All bands from western analysis were quantified for protein
expression with Odyssey Infrared Imaging Software (Li-Cor Biosciences, Lincoln,
Nebraska) to assess integrated intensity (pixel volume) as a measure of optical
39
density. Band density was represented as the ratio of average band intensity (I) of
each sample to the average band intensity of the corresponding β actin control band.

2.2.8 Statistical Analysis
Chi-Square and Fisher’s exact tests were used to compare differences in
Grp78 expression among all groups; if p-values for the overall test were significant
at the 0.05-level, then these were used to analyze pairwise comparisons. Kaplan-
Meier plots and the log-rank test were used to analyze the association of Grp78
expression with time to clinical and/or serum PSA recurrence and survival in the
untreated stage T
3
N
0
M
0
group; the stratified logrank test was used for the
multivariable analyses. Results were considered significant at p<0.05 for 2-sided
analyses.

2.3 Results
2.3.1 Grp78 Expression in Localized Prostate Cancer
Immunohistochemistry was employed to evaluate Grp78 protein levels in
tumors from 164 stage T
3
N
0
M
0
untreated and 27 stage T
3
N
0
M
0
treated prostate
cancer patients. In the untreated group, 120 of 164 cases (73%) demonstrated high
Grp78 expression by percent of cytoplasmic immunoreactivity (>50% stained tumor
cells), as shown in Figure 2-1 and Table 2-1. Of the 27 cases in the treated group,
however, 18 (67%) cases showed high Grp78 percent immunoreactivity (Figure 2-1,
Table 2-1). According to intensity of Grp78 immunoreactivity, 91 of 164 (55%)
40
untreated cases demonstrated moderate to high expression of Grp78 (Figure 2-1A,
Table 2-1). In the treated group, 14 of 27 (52%) tumors showed moderate to high
Grp78 expression (Figure 2-1B,C; Table 2-1). For percent immunoreactivity and
intensity, the differences between the untreated and treated stage T
3
N
0
M
0
groups did
not reach statistical significance (p=0.484, p=0.13).
We identified focal areas of cells showing intense Grp78 immunoreactivity
(≥5%; 3+ intensity) in both the untreated and treated stage T
3
N
0
M
0
cases (Figure 2-
1). In the untreated cases, 36% (59/164) versus 44% of treated cases were classified
as positive for this intense focal immunoreactivity.

2.3.2 Grp78 expression in castration resistant prostate cancer
Of the 28 CRPC tumors immunostained for Grp78, 28 (100%) showed high
Grp78 expression by percent cytoplasmic immunoreactivity (Figure 2-1D, Table 1).
Compared to the untreated and treated stage T
3
N
0
M
0
cases, Grp78 expression
according to percent of immunoreactive tumor cells was significantly increased in
CRPC (p=0.005). This elevation in Grp78 expression remained significant even
when comparing CRPC cases to the untreated and treated groups separately
(p=0.002, p<0.001). When Grp78 expression was examined in CRPC tumors by
intensity of cytoplasmic immunoreactivity, 22 of 28 (79%) cases showed moderate
to high expression (Figure 2-1, Table 2-1). Compared to the stage T
3
N
0
M
0
cases, the
number of tumors showing moderate to high intensity Grp78 expression in the CRPC
group was significantly greater than both the untreated group (p=0.033) and the
41
treated group (p=0.053). Further, when Grp78 expression was examined as a
combined measure of percent of overall immunoreactive tumor cells and intensity
(Grp78 status), Grp78 expression remained significantly elevated in the CRPC group
when compared to both the untreated group (p=0.018) and the treated group
(p=0.037).

2.3.3 In vitro expression of Grp78 corroborates clinical observations
Our cell line model consisted of LNCaP-derived castration resistant C42B
cells and androgen-dependent LNCaP cells grown in medium with fetal calf serum
(FCS) or in androgen-deprived conditions where FCS was replaced with charcoal-
stripped serum (CSS). We found that C42B cells and LNCaP cells maintained in
medium with CSS (androgen-depleted) for 6 days showed prominent cytoplasmic
Grp78 immunoreactivity, as compared to LNCaP cells grown with FCS, which
demonstrated faint cytoplasmic Grp78 immunostaining (Figure 2-1E). Quantitation
of Grp78 cytoplasmic immunoreactivity by AICS II computer imaging of 5
representative areas on each sample slide showed that C42B cells had a mean of
84.0% immunoreactive tumor cells, LNCaP cells grown in CSS for 6 days
demonstrated a mean of 64.2% reactive tumor cells, and LNCaP cells grown in FCS
were found to have an average of 24.5% tumor cells showing cytoplasmic reactivity
to Grp78 antibody. The ACIS II system reported a mean of 1.2% reactive tumor
cells for the negative control LNCaP FCS cells excluding primary antibody.
Intensity of each sample analyzed by ACIS II was also found to be greater in C42B
42
and 6-day hormone-starved LNCaP cells (data not shown) than in LNCaP cells
grown in FCS. As shown in Figure 2A and Table 2, these results were corroborated
by western blot analysis of cell lysates prepared from LNCaP cells grown with FCS,
LNCaP cells grown with CSS for 2, 4, and 6 days, and C42B cells. Comparison of
Grp78 protein levels, expressed as band intensity ratios, showed that Grp78
expression in LNCaP cells was lowest in cells grown with FCS (1.00 standardized
ratio), increased upon androgen starvation for 2 and 4 days (3.63, 2.63 ratios), even
further increased upon 6 days of hormone depletion (8.37 ratio), and was highest in
castration resistant C42B cells (13.94 ratio).

2.3.4 Association of Grp78 expression with Prostate Cancer Recurrence and
Survival
To evaluate Grp78 as a potential marker of prostate cancer progression, we
examined the association of Grp78 expression with cancer recurrence risk and
overall survival in untreated stage T
3
N
0
M
0
patients. Treated cases were excluded
due to potential alterations in Grp78 expression as a result of exposure to hormone
ablation. Untreated cases were stratified by age, PSA level, and Gleason grade. We
examined the associations between Grp78 expression and prostate cancer recurrence
and survival in untreated stage T
3
N
0
M
0
patients (n=164). At 12 years, in the stage
T
3
N
0
M
0
untreated cohort, the probability of remaining recurrence free in cases
expressing low Grp78 (<5% cells with intense immunoreactivity to Grp78) was 64%
versus 54% in those expressing high (>5% cells with intense immunoreactivity)
43
levels of Grp78. Stratification by the standard clinical variables (i.e. a multivariable
analysis, adjusting for age, PSA measurements, and Gleason score (Table 2-3)) of
Grp78 expression in the stage T
3
N
0
M
0
untreated cohort, demonstrated that the risk of
recurring or dying was greater for patients with tumors that expressed high levels of
Grp78 (>5% of tumor cells with 3+ intensity) compared to patients with tumors who
expressed low levels of Grp78 – even after adjusting for these known predictors of
outcome. That is, in Table 2-3, the relative risks which compare patients with high
Grp78 tumors to those with low Grp78 tumors, was not changed substantially, after
stratification. Although these trends did not achieve statistical significance at the
0.05 level, they are consistent across strata - for both recurrence and survival. In
selected subsets, however, Grp78 expression proved to be significant among
particular subsets of patients.
Interestingly, we observed that in the untreated stage T
3
N
0
M
0
patients who
were below the median age of 67 (n=80) at diagnosis, increased Grp78
immunoreactivity (>5% cells expressing high levels of Grp78) was significantly
associated with increased risk of any recurrence (clinical and/or PSA) (p=0.017;
Figure 2-4, Table 2-3). At median follow-up year 12, the probability of remaining
recurrence free in cases expressing low Grp78 was 61% versus 45% in those
expressing high levels of Grp78. The median recurrence-free interval for patients
(n=80) with low versus high Grp78 expression was 14.5 years versus 8.7 years.

44
Figure 2-1. Grp78 expression in prostate cancer. a) Tumor from untreated stage
T
3
N
0
M
0
group showing >50% of tumor cells with moderately (2+) intense Grp78
cytoplasmic immunoreactivity; original magnification x200. b) Tumor from treated
T
3
N
0
M
0
group showing intense (3+) focal Grp78 cytoplasmic immunoreactivity;
original magnification x200. c) Inset from (b); subpopulations of prostate cancer
cells demonstrate intense Grp78 cytoplasmic immunoreactivity (arrows). d) Tumor
from castration resistant group showing high (3+) intensity cytoplasmic
immunoreactivity; original magnification x200. e) Immunostaining of Grp78 in
androgen dependent LNCaP cells grown in FCS, LNCaP cells grown in androgen-
depleted medium for six days (6dCSS), and castration resistant C42B cells. ACIS II-
assisted computer imaging analysis shows that C42B (84% tumor cells reactive) and
LNCaP cells grown in CSS for 6 days (64.2% tumor cells reactive) showed higher
Grp78 cytoplasmic immunoreactivity than cells grown in FCS (24.5% tumor cells
reactive); percentages given as mean value of 5 representative areas ± standard
deviation. Negative control with no primary antibody shown in the left panel;
original magnification x100; inset magnification x200.

45
Figure 2-1. Grp78 expression in prostate cancer.

46
Table 2-1. Grp78 expression (immunoreactivity) in untreated T
3
N
0
M
0,
treated
T
3
N
0
M
0
, and castration resistant prostate cancer.

47
Figure 2-2. Grp78 expression in prostate cancer cells during brief and
prolonged androgen starvation. Western blot analysis for Grp78 expression in
LNCaP cells grown in fetal calf serum (FCS) and charcoal-stripped serum (CSS, for
two, four, and six days) and castration resistant C42B cells; β actin loading control
shown in the lower panel; numbers represent the ratio of sample band density to β
actin band density, using the lowest ratio (LNCaP FCS) as the reference point of
1.00.

48
Table 2-2. Quantification of protein expression from western blot analysis of
Grp78. All measurements taken as average band density with data units of optical
density (OD). Standardized ratio calculated using lowest ratio I/βI as reference point
of 1.00.

49
Figure 2-3. Probability of recurrence-free (clinical and/or PSA) status in 164
patients with stage T
3
N
0
M
0
prostate cancer, based on levels of Grp78
immunoreactivity. Untreated stage T
3
N
0
M
0
patients demonstrated greater
probability of prostate cancer recurrence with higher Grp78 expression. Tick marks
represent patients with no evidence of disease at last follow-up. The P value was
obtained using the log-rank test.

50
Table 2-3. Grp78 expression and recurrence-free or overall survival of patients
with untreated stage T
3
N
0
M
0
tumors. Percent (<5 or ≥5%) of tumor cells with 3+
intense immunoreactivity represents Grp78 expression. Hazard ratios were
calculated as a measure of relative risk (RR). Recurrence includes clinical and/or
PSA recurrence. PSA values were not available for 54 patients who were excluded
from PSA stratified analyses.

Abbreviation: NA, not available.
*Percentage (<5% or ≥5%) of tumor cells with 3+ intense immunoreactivity represents Grp78 expression.
†Recurrence includes clinical and/or PSA recurrence.
‡Hazard ratios were calculated as a measure of relative risk.
§Number of patients with ≥5% tumor cells with 3+ Grp78/number of patients with <5% tumor cells with 3+ Grp78.
‖ PSA values were not available for 54 patients who were excluded from PSA-stratified analyses.

51
Figure 2-4. Probability of recurrence-free (clinical and/or PSA) status in 80
patients with stage T
3
N
0
M
0
prostate cancer stratified by median age, based on
levels of Grp78 immunoreactivity. Untreated stage T
3
N
0
M
0
patients were stratified
by age, where patients under the cohort median age of 67 years (n=80) demonstrated
greater probability of prostate cancer recurrence with higher Gr78 expression. Tick
marks represent patients with no evidence of disease at last follow-up. The P value
was obtained using the log-rank test.

52
2.4 Discussion
Based on previous studies identifying the role of Grp78 in cell survival and
cancer progression [Lee 2001, Mintz 2003, Fernandez 2000], we hypothesized that
expression of Grp78 is correlated with prostate cancer progression and the
development of CRPC. In the current study, we examined the expression of Grp78
in 3 cohorts of prostate cancer patients, uniquely designed to represent successive
stages in the development of CRPC. This included men who were not exposed to
hormone therapy, men who were exposed and considered responsive to hormone
therapy, and men who were exposed and resistant to hormone therapy. Our results
suggest that Grp78 expression is upregulated during the transition from localized
prostate cancer to metastatic CRPC. Thus, it is possible that overexpression of
Grp78 may confer resistance to apoptosis in stress conditions such as anti-androgen
therapy, and may be an integral component of castration resistant growth.
Clonal selection has been implicated in the development of CRPC [Craft
1999]. This is corroborated by identification of focal areas showing intense Grp78
immunoreactivity (≥5%; 3+ intensity) in both the untreated and treated stage T
3
N
0
M
0

cases (Figure 2-1B, C) where a greater percentage of treated cases (44%) versus
untreated cases (36%) were classified as positive for this intense focal
immunoreactivity. We have also provided corroborating in vitro evidence, using our
cell line model which mimics the clinical development of CRPC, of increased Grp78
expression in castration resistant growth, further suggesting the feasibility of Grp78
as a novel therapeutic target for prostate cancer.
53
It has been postulated that Grp78 induction occurs in response to cellular
stress, is involved in escape from apoptosis, and contributes to drug resistance
[Reddy 2003, Rao 2002, Miyake 2000, Shen 1987]. Recent evidence also suggests
that Grp78 may contribute to the metastatic potential of prostate cancer cells via α-2-
macroglobulin-mediated signaling [Misra 2005]. Our findings accordingly showed
significantly higher expression of Grp78 in metastatic CRPC when compared to
localized stage T
3
N
0
M
0
prostate cancer. This result is also consistent with those
reported by Mintz et al [Mintz 2003] that show strong Grp78 epitope
immunoreactivity in bone marrow metastases from castration resistant patients
versus weak immunostaining in normal prostate, and higher serum immunoreactivity
to Grp78 in CRPC as compared to locally advanced prostate cancer.
Interestingly, we found that Grp78 expression is relatively unchanged in
androgen responsive tumors on initial exposure to anti-androgen therapy. However,
we identified focal areas of tumor cells showing intense Grp78 immunoreactivity in
both the untreated and treated stage T
3
N
0
M
0
cases, where a greater percentage of
treated cases versus untreated cases were classified as positive for this intense focal
immunoreactivity. Craft et al has previously established in their xenograft prostate
tumor model the existence of castration resistant cells, even before hormone ablation
[Craft 1999]. It is possible that the focal areas of prostate tumor, which strongly
express Grp78, are comprised of clones which have been conferred a survival
advantage by overexpressing Grp78, which may potentially develop into the more
homogeneous staining cells in castration resistant prostate cancer that demonstrate
54
strong Grp78 immunoreactivity. Thus, our results are suggestive of clonal selection,
whereby androgen withdrawal allows for selective survival and proliferation of
castration resistant cells that existed upon initiation of therapy [So 2005].
Using our previously established cell line model of CRPC development [Shi
2006], we demonstrated the occurrence of increased Grp78 expression in conditions
of androgen deprivation, and even greater Grp78 expression in established castration
resistant C42B cells. Further, the application of quantitative methods such as
densitometry for western blot analysis and ACIS II automated image analysis for
IHC allowed for objective assessment of relative Grp78 levels. Given the subjective
nature of immunohistochemical reactivity assessment, results from the novel ACIS II
system showing increased Grp78 in castration resistant growth as compared to
androgen dependent growth provided numerical validation of our clinical findings.
In this study, we observed that increased Grp78 expression in prostate tumors
is associated with greater relative risk of recurrence and worse overall survival. This
pattern was still observed following stratified analyses. Further, Grp78 expression
may have important prognostic value in distinct subsets of patients, particularly in
those for whom favorable prognosis is indicated by low PSA levels or low Gleason
grade, as suggested by Table 2-3. Our results also suggest that increased Grp78
expression may be predictive of prostate cancer recurrence among patients who are
diagnosed at relatively earlier age. These and further confirmatory studies may
potentially indicate more aggressive treatment in patients who demonstrate high
55
levels of Grp78, particular for younger patients who may be better able to undergo
such therapies.
A number of other stress-induced proteins have been implicated in the
development of CRPC. Heat shock protein Hsp70, which shares 60% homology
with Grp78, and other stress response proteins, such as Hsp27 and Hsp90, appear to
contribute substantially to prostate cancer progression and the development of
CRPC, where suppression of these proteins is inhibitory to castration resistant
growth [Munro 1986, Zhao 2004, Rocchi 2004, Cornford 2000]. This is indicative
of the potential role of Grp78 in CRPC development and as an additional therapeutic
target which, if inhibited, may allow cells to become susceptible to environmental
stresses.
A significant clinical problem in prostate cancer is that patients who are
initially responsive to androgen ablation therapy often develop CRPC, despite recent
advances in cancer therapy. One of the most common molecular defects associated
with CRPC is deregulated androgen receptor (AR) pathway, resulting in
constitutively active cell proliferative machinery. A better understanding of the
complex contributing molecular mechanisms underlying the development of CRPC
is necessary so that the most appropriate therapeutic targets may be identified.
Feldman et al proposed five molecular pathways through which prostate cancer cells
may undergo transition from androgen dependent to castration resistant growth
[Feldman 2001]. Among these pathways are mechanisms which result in
downstream AR activation, or those in which alternative signals are upregulated and
56
bypass the necessity to activate AR-regulated transcription. Although the precise
role of Grp78 in the development of CRPC is unclear, increased Grp78 expression
may confer survival advantage through a number of prospective courses, including
its molecular chaperone functions in assisting proper protein folding, inhibition of
the apoptotic pathway, and contribution to growth and invasive potential of prostate
cancer cells via cell surface receptor functions [Reddy 2003, Shen 1987, Li 2006,
Arap 2004]. The intrinsic functions of Grp78 indicate a potential role in the AR
bypass pathway of CRPC development, although we believe that Grp78 signaling
may contribute to a multitude of molecular mechanisms resulting in castration
resistant growth. Our studies suggest that upregulation of Grp78 stress response
protein plays a role in the progression of localized prostate cancer to CRPC, and
promote Grp78 as a potential prognostic marker and therapeutic target for prostate
cancer.

57
Chapter Three: Expression and Cellular Localization of Survivin is Associated
with Prostate Cancer Aggressiveness, Clinical Outcome, and the Development
of Castration Resistance

3.1 Introduction
Despite recent advances in cancer therapies and current scientific findings
identifying molecular signals which contribute to prostate cancer progression,
resistance to androgen ablation treatments persists as the predominant challenge in
the management of advanced prostate cancer. Androgen dependent prostate cancer
is characterized by the ability of cancer cells to undergo apoptosis in response to
androgen withdrawal. The transition to castration resistant prostate cancer (CRPC)
requires the survival of tumor cells in such conditions, which may be attributed to a
number of biomolecular processes resulting in the evasion of apoptosis. One
potential cellular survival mechanism in CRPC is through upregulation survival
pathways which bypass the need for androgen receptor (AR) activation and allow
cell survival under adverse conditions such as androgen withdrawal.
Survivin, a 16.5-kDa protein, is the smallest member of the inhibitor of apoptosis
(IAP) family [Ambrosini 1997] and has been identified as a nuclear and cytosolic protein
[Fortugno 2002]. While Survivin is strongly expressed in embryonic development, it is
generally undetected in terminally differentiated normal tissue [Adida 1998, Ambrosini
1997, Kobayashi 1999]. Distinct functions have been identified for Survivin, including
those involved in mitotic regulation and cell death inhibition. Survivin expression and
58
activity is crucial for proper mitotic progression [Uren 2000, Kallio 2001]. Further,
mechanistic evidence of an anti-apoptotic role has been reported by Shin et al, who
describe the direct inhibition of caspases 3 and 7 by Survivin [Shin 2001].
The intrinsic functions of Survivin demonstrate a prospective role in cancer cell
survival. Overexpression of Survivin has previously been reported in a number of
hormonally influenced cancers, including cancers of the breast, uterus, and ovaries
[Tanaka 2000, Saitoh 1999, Yoshida 2001]. Further, cytoplasmic expression of Survivin
has previously been associated with poorer prognosis in a number of tumor
malignancies, including neuroblastoma, colorectal, and bladder cancers [Adida 1998,
Kawasaki 1998, Swana 1999], while nuclear expression of Survivin has been correlated
with favorable prognosis in gastric cancer [Okada 2001]. It has recently been
demonstrated that Survivin promotes resistance to antiandrogen treatment in prostate
cancer, both in vitro and in vivo [Zhang 2005]. Increased expression of the Survivin
gene and protein has also been seen in clinical prostate cancer samples, as compared to
control tissue [Kishi 2004, Kaur 2004, Shariat 2004]. Fromont et al has also shown
significantly elevated Survivin gene expression in CRPC compared to localized prostate
cancer [Fromont 2005], in a study which included a cohort of 33 stage T
2
and T
3

patients, and 13 CRPC patients. Because it is the action of Survivin protein which
mediates the regulation of mitosis and programmed cell death, and possible involvement
in resistance to androgen ablation therapy and progression of prostate cancer cells to an
androgen independent state may be attributed to these functions, it follows that
expression levels of Survivin should be examined in a clinical cohort designed to assess
59
molecular alterations occurring from localized prostate cancer to the development of
CRPC, after antiandrogen therapies have proved unsuccessful.
As stated above, Survivin nuclear expression has been correlated with
favorable prognosis in cancer, while cytoplasmic expression has been predictive of
poorer prognosis. These findings suggest that in addition to increased expression,
subcellular distribution of Survivin may be significant in cancer progression. We
were interested in determining if nuclear and cytoplasmic Survivin expression is
altered during the progression of prostate cancer from localized disease to CRPC.
As Survivin involvement has previously been shown in resistance to antiandrogen
therapy in prostate cancer cells [Zhang 2005], we also aimed to confirm these
findings in our cohort of human prostate cancer samples to establish clinical
relevance of Survivin expression in resistance to antiandrogen treatment.

3.2 Materials and Methods
3.2.1 Patient Population
The recruitment and studies of patients are as described in Chapter 2.2.1.
The stage T
3
N
0
M
0
untreated group included 125 patients who were not exposed to
pre-operative androgen ablation therapy. The group of 28 men comprising the stage
T
3
N
0
M
0
treated group had received pre-operative neoadjuvant androgen ablation
therapy. All stage T
3
N
0
M
0
patients were considered responsive to anti-androgen
therapy [Fradet 1996]. Tumor samples were obtained from 32 patients with CRPC
who underwent hormone ablation via orchiectomy and systemic hormone therapy
60
but continued to demonstrate rising prostate specific antigen (PSA). Between 1990
and 1992, these men underwent transurethral resection to relieve urinary obstruction
at Ruhr University, Bochum, Germany. All tumor grading was in concordance with
the Gleason system [Gleason 1977].

3.2.2 Patient Followup
Evaluations of all patients were as described in Chapter 2.2.2. Median
follow-up in the untreated group of 125 patients was 12.3 years with a range of 1.6 to
20 years. Median age was 66 years, ranging from 47 to 81 years. For clinical
outcome studies, treated cases were excluded due to ranges in treatment and
therefore potential alterations in Survivin expression as a result of exposure to
hormone ablation.

3.2.3 Immunohistochemistry (IHC)
Immunohistochemistry was performed as described in Chapter 2.2.3. Briefly,
the slides were blocked with normal horse serum for 20 minutes, and incubated for 1
hour with anti-Survivin rabbit polyclonal antibody (Neomarkers) at a 1:300 dilution
in phosphate-buffered saline. Incubation with biotinylated horse anti-rabbit
secondary antibody at a 1:200 dilution was followed by avidin-biotin-conjugate
(ABC, Vector Laboratories, Inc., Burlingame, CA). Chromogen of 0.03%
diaminobenzidine was then applied, with hematoxylin counterstaining. Negative
61
controls consisting of diluent with no antibody, and positive prostate cancer controls
with heterogeneous immunoreactivity were used in all experiments.
Cultured prostate cancer cells (LNCaP, C42B) were harvested, cytospun on
poly-L-lysine coated slides at 250,000 cells per slide, and formalin-fixed for 30
minutes. Antigen retrieval was performed using citrate buffer (pH=6) and
microwaving in a pressure cooker for 30 minutes [Shi 1997], followed by cooling at
room temperature for 15 minutes. Subsequent steps in IHC protocol follow as
described above.

3.2.4 Immunoreactivity Assessment of Clinical Samples
All slides were interpreted by a pathologist (MA), who was blinded to all
outcome data. Tumor scores were categorized based on 2 criteria: 1) percent of
tumor cells demonstrating nuclear or cytoplasmic immunoreactivity and 2) intensity
of nuclear or cytoplasmic immunostaining. For assessment according to percent of
tumor cells showing nuclear or cytoplasmic reactivity, tumors were classified as
negative for Survivin expression (<10%; 0), as having low Survivin expression (10-
30%; 1), moderate Survivin expression (31-50%; 2), or high Survivin expression
(>50%; 3). For intensity of nuclear or cytoplasmic immunoreactivity, tumors were
classified as having low Survivin expression (1+), moderate Survivin expression
(2+), or high Survivin expression (3+). Combined Survivin status (percent of
immunoreactive tumor cells and intensity of Survivin immunoreactivity) was
assigned as negative to cases with a combined factor of 3 (percent x intensity). All
62
other cases were assigned positive Survivin status. Due to the heterogeneity of
Survivin immunoreactivity, scoring corresponds with an overall evaluation of the
entire tissue section. Ten high power fields (hpf) were evaluated per slide for both
nuclear (magnification x400) and cytoplasmic (magnification x200) Survivin
immunoreactivity. For assessment of nuclear immunostaining, a total of 1000 tumor
cells (100 cells per hpf) were counted in each sample.

3.2.5 Cell Culture
LNCaP and C42B cells were cultured as described in Chapter 2.2.5.

3.2.6 Automated Cellular Imaging
Immunostaining and evaluation of immunostained cell lines were carried out
in triplicate, where immunoreactivity was assessed as described in Chapter 2.2.6.

3.2.7 Western Blot Analysis
For western blot analysis, cell lysates from LNCaP and C42B cells were
prepared by lysing in 1mL ice-cold RIPA buffer. Equal amounts of total protein
from each sample were subjected to SDS-PAGE in a 4-20% Tris-HCl gel (NuSep,
Sydney, Australia). Following electrophoresis, the proteins were transferred to a
pure nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). The
membrane was then incubated in Odyssey Blocking Buffer (Li-Cor Biosciences,
Lincoln, Nebraska), followed by overnight incubation with primary mouse
63
monoclonal anti-Survivin antibody (1:500 dilution; Santa Cruz Biotechnology Inc,
Santa Cruz, CA). Signal detection was performed using Alexa Fluor 680 goat anti-
rabbit antibody (Molecular Probes, Eugene, Oregon) and subsequent scanning of the
membrane by the Odyssey Infrared Imager (Model 9120, Li-Cor Biosciences,
Lincoln, Nebraska). All bands from western analysis were quantified for protein
expression with Odyssey Infrared Imaging Software (Li-Cor Biosciences, Lincoln,
Nebraska) to assess integrated intensity (pixel volume) as a measure of optical
density. Band density was represented as the ratio of average band intensity (I) of
each sample to the average band intensity of the corresponding β actin control band.

3.2.8 Cellular Compartmentalization Assay
Cultured prostate cancer cells (LNCaP, C42B) were pelleted, separated into
nuclear and cytoplasmic fractions using the NE-PER Nuclear and Cytoplasmic
Extractions Kit according to manufacturer’s protocol (Pierce Biotechnology,
Rockford, IL), and subsequently analyzed by western blot using anti-Survivin
primary antibody and anti β actin primary antibody. Briefly, 20-40 μg cell pellets
were resuspended in 200 μl cytoplasmic extraction buffer and incubated on ice.
Following centrifugation, the supernatant was collected as the cytoplasmic fraction,
and the remaining pellet was resuspended in nuclear extraction buffer. The
suspension was centrifuged, with the remaining supernatant collected as the nuclear
fraction.

64
3.2.9 Statistical Analysis
Chi-Square and Fisher’s exact tests were used as described in Chapter 2.2.8.

3.3 Results
3.3.1 Expression of Cytoplasmic Survivin in Localized Prostate Cancer
To evaluate Survivin protein levels, we performed immunohistochemical
analysis on tumors from 125 stage T
3
N
0
M
0
untreated and 28 stage T
3
N
0
M
0
treated
prostate cancer patients. In the untreated group, 95 of 125 cases (76%) demonstrated
high Survivin expression by percent of cytoplasmic immunoreactivity (>30% stained
tumor cells; Figure 3-1). Of the 28 cases in the treated group, however, 24 (85%)
cases showed high Survivin percent cytoplasmic immunoreactivity (Figure 3-1).
According to intensity of Survivin immunoreactivity for cytoplasmic staining, 44 of
125 (35%) untreated cases demonstrated moderate to high expression of Survivin
(Figure 3-1, Table 3-1). In the treated group, 11 of 28 (39%) tumors showed
moderate to high Survivin cytoplasmic expression (Figure 3-1, Table 3-1). For
percent immunoreactivity and intensity of cytoplasmic Survivin, the differences
between the untreated and treated groups did not reach statistical significance
(p=0.2364, p=0.7348). We combined the measurements of percent of
immunoreactive tumor cells and intensity of immunoreactivity for cytoplasmic
localized Survivin and termed this classification as cytoplasmic Survivin status. For
the untreated stage T
3
N
0
M
0
cases, cytoplasmic Survivin status was positive in 83 of
125 (66%) of tumors, and in 22 of 28 (79%) of the treated stage T
3
N
0
M
0
cases, and
65
the difference between untreated and treated stage T
3
N
0
M
0
cases for cytoplasmic
Survivin status was not statistically significant (Table 3-1, p=0.2096).

3.3.2 Expression of Nuclear Survivin in Localized Prostate Cancer
In the untreated group, cases showed high Survivin expression by percent of
nuclear reactivity in 25 of 125, or 20% of the cases (Figure 3-1). Of the 28 cases in
the treated group, 2 of 28 cases (7%) had high Survivin nuclear reactivity by percent
of tumor cells staining positive (Figure 3-1). There were 32 of 125 (26%) untreated
cases demonstrating moderate to high intensity nuclear expression of Survivin
(Figure 3-1, Table 3-2). In the treated group, 4 of 28 (14%) cases demonstrated
moderate to high Survivin nuclear immunoreactivity by intensity (Figure 3-1, Table
3-2). For percent immunoreactivity and intensity of nuclear Survivin, the differences
between the untreated and treated groups did not reach statistical significance
(p=0.1812, p=0.4714). As we did for cytoplasmic Survivin, we combined the
measurements of percent of immunoreactive tumor cells and intensity of
immunoreactivity for nuclear localized Survivin and termed this classification as
nuclear Survivin status. For the untreated stage T
3
N
0
M
0
cases, nuclear Survivin
status was positive in 33 of 125 (27%) of tumors, and was also positive in 2 of 28
(7%) of the treated stage T
3
N
0
M
0
cases (Table 3-2). According to nuclear status,
nuclear Survivin was significantly decreased in treated stage T
3
N
0
M
0
tumors as
compared to untreated stage T
3
N
0
M
0
tumors (p=0.0283).

66
3.3.3 Survivin Cytoplasmic Expression in Castration Resistant Prostate Cancer
There were 32 castration resistant tumors immunostained for Survivin, of
which 29 (91%) showed high cytoplasmic Survivin expression by percent of tumor
cells with immunoreactivity (Figure 3-1). When Survivin expression was examined
in CRPC tumors by intensity of cytoplasmic immunoreactivity, 25 of 32 (78%) cases
showed moderate to high expression (Figure 3-1, Table 3-1). Compared to the stage
T
3
N
0
M
0
cases, the number of tumors showing moderate to high intensity Survivin
expression in the CRPC group was significantly greater than both the untreated
group (p<0.0001) and the treated group (p=0.002), while the difference in CRPC
cases versus untreated and treated cases by percent of cytoplasmic immunoreactivity
did not reach statistical significance (p=0.2239, p=0.9151). Further, when
cytoplasmic Survivin expression was examined as a combined measure of percent of
overall immunoreactive tumor cells and intensity (cytoplasmic Survivin status),
Survivin expression was significantly elevated in the CRPC group when compared to
both the untreated group (p=0.0006) and the treated group (p=0.028).

3.3.4 Survivin Nuclear Expression in Castration Resistant Prostate Cancer
Of the 32 CRPC tumors immunostained for Survivin, 4 (13%) showed high
Survivin expression by percent nuclear immunoreactivity (Figure 3-1). Compared to
the untreated and treated stage T
3
N
0
M
0
cases, nuclear Survivin expression according
to percent of immunoreactive tumor cells was not statistically significant (p=0.7534,
p=0.3834). The elevation in Survivin expression, however, was strongly significant
67
in CRPC tumors when nuclear Survivin expression was examined by intensity of
nuclear immunoreactivity. There were 23 of 32 (72%) CRPC cases which showed
moderate to high nuclear Survivin expression (Figure 3-1, Table 3-2). Compared to
the stage T
3
N
0
M
0
cases, the number of tumors showing moderate to high intensity
nuclear Survivin expression in the CRPC group was significantly greater than both
the untreated group (p<0.0001) and the treated group (p=0.0001). When overall
nuclear Survivin status was assessed, as a combined measure of percent of overall
immunoreactive tumor cells and intensity, Survivin expression was significantly
elevated in the CRPC group when compared to the treated group (p=0.036) but not
the untreated group (p=0.844).

3.3.5 In Vitro Observations of Survivin Expression Support Clinical Findings
Our cell line model consisted of castration-resistant C42B cells and
androgen-dependent LNCaP cells grown in medium with fetal calf serum (LNCaP
FCS) or in androgen-deprived conditions where FCS was replaced with charcoal-
stripped serum (LNCaP 6dCSS). We found that LNCaP cells grown in FCS
demonstrated moderate cytoplasmic Survivin immunoreactivity, and similarly
LNCaP cells maintained in medium with CSS for 6 days showed moderate
cytoplasmic Survivin immunoreactivity (Figure 3-2A). Castration resistant C42B
cells, however, demonstrated strong cytoplasmic immunoreactivity, as compared to
LNCaP FCS or LNCaP 6dCSS cells (Figure 3-2A). For nuclear Survivin
immunoreactivity, LNCaP FCS showed weak to moderate nuclear immunostaining,
68
while LNCaP 6dCSS cells demonstrated little or no nuclear Survivin reactivity. We
observed the strongest nuclear Survivin immunoreactivity in C42B cells. As shown
in Figure 3-2B, these results of immunohistochemical analysis on cytospun cells
were confirmed by Western blot analysis of whole cell lysates prepared from LNCaP
FCS cells, LNCaP 6dCSS cells, and C42B cells. Comparison of Survivin protein
levels, expressed as band intensity ratios, showed that compared to Survivin
expression in LNCaP cells grown with FCS (2.30 ratio), Survivin expression was
decreased upon prolonged androgen starvation for 6 days (standardized ratio 1.00),
and subsequently increased and was highest in castration resistant C42B cells (4.30
ratio).
Western blot analysis of nuclear and cytoplasmic fractions of LNCaP and
C42B cells also supported clinical findings and results found by immunostaining
cytospun prostate cancer cells. For cytoplasmic fractions, we found that Survivin
expression decreased slightly in androgen dependent LNCaP cells upon androgen
withdrawal and subsequently increased in castration resistant C24B cells (Figure 3-
3). Western analysis of nuclear fractions showed that nuclear Survivin decreased
upon androgen withdrawal treatment, and subsequently increased in castration
resistant C42B cells (Figure 3-3). Figures 3-1, 3-2, and 3-3 demonstrate that overall
Survivin expression levels had similar trends upon androgen starvation and in
castration resistant growth in clinical samples, whole cell lysates from prostate
cancer cells, and compartmentalized fractions of prostate cancer cells.

69
3.3.6 Association of Nuclear and Cytoplasmic Survivin Expression with Prostate
Cancer Recurrence and Survival
To evaluate Survivin as a potential marker of prostate cancer progression, we
examined the association of both nuclear and cytoplasmic Survivin expression with
prostate cancer recurrence risk and overall survival in 125 untreated stage T
3
N
0
M
0

patients. Three untreated stage T
3
N
0
M
0
cases were excluded due to incomplete
follow up. Treated cases were excluded due to potential alterations in Survivin
expression as a result of exposure to hormone ablation. Untreated cases were
stratified by age, PSA level, and Gleason grade.
At assessment of nuclear Survivin immunoreactivity with clinical outcome,
we found that the probability of remaining recurrence free (clinical and/or PSA) in
cases expressing low nuclear Survivin (according to nuclear Survivin status;
combined measure of intensity and percent of immunoreactive tumor cells) was
significantly greater than in cases expressing high levels of nuclear Survivin
according to nuclear Survivin status (p=0.01; Figure 3-4). At median follow-up of
12 years in our stage T
3
N
0
M
0
untreated cohort, the probability of remaining
recurrence free in cases expressing low nuclear Survivin was 72% versus 46% in
those expressing high levels of Survivin. When examining the measures of nuclear
Survivin expression separately, there were trends towards significance of association
with recurrence for both intensity (p=0.08) and percent of immunoreactive tumors
cells (p=0.06; Figure 3-5). The probability for remaining recurrence free for cases
expressing the lowest versus highest levels of Survivin according to intensity was
70
70% versus 42%, and was 68% versus 40% for cases demonstrating the lowest
versus highest expression of Survivin according to percent of tumor cells with
nuclear immunoreactivity. Correlations between nuclear Survivin expression and
overall survival did not reach statistical significance.
For analysis of cytoplasmic Survivin, we determined that in the untreated
stage T
3
N
0
M
0
patients, those with highest Survivin immunoreactivity by intensity
(3+) had over 2 times the relative risk of prostate cancer recurrence compared to
those who had no Survivin immunoreactivity (0; data not shown). While the overall
association of Survivin cytoplasmic staining intensity with recurrence did not reach
statistical significance (p=0.1446), at median follow-up year 12, the probability of
remaining recurrence free in cases expressing lowest Survivin according to intensity
was 100% versus 30% in those expressing highest levels of Survivin. The median
recurrence-free interval for patients with low versus high Survivin expression by
intensity was 16.9 years versus 9.7 years. When examining cytoplasmic Survivin
expression by percent of immunoreactive tumor cells or overall cytoplasmic Survivin
status, we did not find statistically significant correlations with prostate cancer
recurrence or overall survival.
We looked at the association between Survivin expression and tumor grade in
stage T
3
N
0
M
0
untreated cases and found that Survivin expression is significantly
correlated with Gleason score. According to cytoplasmic intensity, higher Survivin
expression was significantly associated with higher Gleason score in stage T
3
N
0
M
0
tumors (p=0.01). Upon assessment of nuclear intensity of Survivin with tumor
71
grade, we found that higher Survivin expression was strongly associated with higher
Gleason score (p=0.006).

3.3.7 Survivin Expression Shares Coordinate Expression with Glucose-
Regulated Protein 78 (Grp78)
To further elucidate possible mechanisms by which prostate cancer cell
growth is regulated, we examined the association between Survivin expression and
Grp78 expression in stage untreated stage T
3
N
0
M
0
patients. We found that Survivin
levels and Grp78 levels were significantly associated in the untreated stage T
3
N
0
M
0

patients, according to intensity of cytoplasmic Survivin (p=0.0124). When looking
at overall cytoplasmic Survivin status (combined intensity and percent of
immunoreactive tumors cells) and its association with Grp78 status, this correlation
became strongly significant (p=0.0068). Nuclear Survivin expression was not
associated with Grp78 expression by nuclear intensity or percent of tumor cells with
nuclear Survivin immunoreactivity (p=0.4380, p=0.2137).

72
Figure 3-1. Survivin expression in prostate cancer. A,B) Tumors from untreated
T
3
N
0
M
0
group showing >50% of tumor cells with moderately (2+) intense Survivin
cytoplasmic immunoreactivity; original magnification x200; C,D) Tumors from
untreated group showing intense (3+) Survivin nuclear immunoreactivity and
negative or weak cytoplasmic reactivity; original magnification x200; E,F) Tumors
from untreated T
3
N
0
M
0
group showing intense (3+) Survivin nuclear
immunoreactivity and moderate to strong cytoplasmic immunostaining; G) Tumor
from treated stage T
3
N
0
M
0
group showing negative nuclear Survivin
immunoreactivity and moderate (2+) cytoplasmic reactivity; H, I) Tumors from
castration resistant group showing negative nuclear (H) and strongly intense nuclear
(I) immunostaining with high (3+) intensity cytoplasmic immunoreactivity; original
magnification x200.

73
Table 3-1. Survivin cytoplasmic expression (immunoreactivity) in untreated
T
3
N
0
M
0
, treated T
3
N
0
M
0
, and castration resistant prostate cancer (CRPC)

74
Table 3-2. Survivin nuclear expression (immunoreactivity) in untreated
T
3
N
0
M
0
, treated T
3
N
0
M
0
, and castration resistant prostate cancer (CRPC)

75
Figure 3-2. Survivin expression in prostate cancer cell lines. A)
Immunohistochemical analysis of nuclear and cytoplasmic Survivin in LNCaP cells
grown in FCS, LNCaP cells grown in androgen-depleted medium for six days
(6dCSS), and C42B cells; numbers represent brown pixel intensity ± standard
deviation, as measured by automated cellular imaging (ACIS II) B) Western blot
analysis for Survivin expression in LNCaP cells grown in fetal calf serum (FCS) and
charcoal-stripped serum (CSS, for six days) and castration resistant C42B cells; b
actin loading control shown in the lower panel; numbers represent the ratio of sample
band intensity to beta actin band intensity, as quantified by densitometry, using the
lowest ratio (LNCaP 6dCSS) as the reference point of 1.00.

76
Figure 3-3. Survivin localization in prostate cancer cells upon androgen
deprivation and castration resistant growth. Western blot analysis for Survivin
expression in nuclear and cytoplasmic compartments of LNCaP cells grown in fetal
calf serum (FCS) and charcoal-stripped serum (CSS, for six days) and castration
resistant C42B cells; b actin loading control shown in the lower panel; numbers
represent the ratio of sample band intensity to beta actin band intensity, as quantified
by densitometry, using the lowest ratio (LNCaP 6dCSS) as the reference point of
1.00.

77
Figure 3-4. Probability of recurrence-free (clinical and/or PSA) status in 122
patients with stage T
3
N
0
M
0
prostate cancer, based on combined intensity and
percent of tumor cells (status) with nuclear Survivin immunoreactivity.
Untreated stage T
3
N
0
M
0
patients demonstrated greater probability of prostate cancer
recurrence with higher nuclear Survivin expression. Tick marks represent patients
with no evidence of disease at last follow-up. The P value was obtained using the
log-rank test.

78
Figure 3-5. Probability of recurrence-free (clinical and/or PSA) status in 122
patients with stage T
3
N
0
M
0
prostate cancer, based on percent of tumor cells
with nuclear Survivin immunoreactivity. Untreated stage T
3
N
0
M
0
patients
demonstrated greater probability of prostate cancer recurrence with higher nuclear
Survivin expression. Tick marks represent patients with no evidence of disease at
last follow-up. The P value was obtained using the log-rank test

79
3.4 Discussion
Because increased expression of Survivin has previously been reported in a
number of hormonally influenced cancers, including cancers of the breast, uterus,
and ovaries [Tanaka 2000, Saitoh 1999, Yoshida 2001], we were interested in
examining Survivin expression in prostate cancer, and its potential role in clinical
outcome and the development of castration resistance. Additionally, other groups
identified the significance of cytoplasmic expression of Survivin with poorer
prognosis in a number of tumor malignancies, including neuroblastoma, colorectal,
and bladder cancers [Adida 1998, Kawasaki 1998, Swana 1999], while nuclear
expression of Survivin has been correlated with favorable prognosis in gastric cancer
[Okada 2001]. We were therefore interested in assessing whether Survivin cellular
localization was a significant factor in evaluating the role of Survivin in prostate
cancer. In this study, we examined the expression of Survivin in three distinct
cohorts of prostate cancer patients, designed to represent the successive stages in the
development of CRPC. These cohorts included men with stage T
3
N
0
M
0
disease who
were not exposed to androgen ablation therapy, men with stage T
3
N
0
M
0
disease who
have been exposed to androgen ablation and are considered responsive to treatment,
and men with CRPC. Our results suggest that Survivin is upregulated during the
development of CRPC from localized prostate cancer, which was demonstrated in
clinical prostate cancer and further corroborated using an in vitro model which
mimics the clinical development of castration resistance. Interestingly, we found
that while cytoplasmic Survivin levels remained relatively similar upon androgen
80
withdrawal before increasing in castration resistant growth, nuclear Survivin
dramatically decreased upon androgen ablation therapy, followed by a significant
increase in CRPC. These results are in agreement with previous studies reporting
strong Survivin expression in LNCaP prostate cancer cells, which is decreased upon
initial antiandrogen therapy, and can subsequently increase upon AR-independent
IGFR1 signaling via the AKT pathway, providing a mechanism through which
prostate cancer cells may develop resistance to androgen ablation [Zhang 2005].
Mahotka et al previously identified differential subcellular localization of
functionally divergent splice variants [Mahotka 2002]. Survivin-ΔEx3, a Survivin
splice variant which lacks exon 3, was shown to be the only variant of Survivin
demonstrating preferential localization in the nucleus, and has been shown to have
antiapoptotic function [Mahotka 2002]. It has also been suggested that nuclear-
cytoplasmic shuttling of Survivin may be important in the regulation of Survivin
function [Rodriguez 2002]. Because our study suggests that Survivin expression in
the nucleus is significantly different upon androgen ablation treatment and in the
development of castration resistance, it is possible that the differential localization of
a particular splice variant shuttling between the nuclear and cytoplasmic
compartments is critical during the development of castration resistance.
In accordance with recent studies which demonstrated the association
between Survivin levels and biologically aggressive prostate cancer [Kishi 2004,
Shariat 2004], we found that increased expression of Survivin was associated with
tumor aggressiveness. Unique to this study, however, was the finding that increases
81
in both nuclear and cytoplasmic Survivin correlated with higher tumor grade, and
that the association with disease aggressiveness with nuclear Survivin was
particularly strong. Additionally, we found that nuclear, but not cytoplasmic,
Survivin expression was significantly associated with prostate cancer recurrence in
stage T
3
N
0
M
0
patients. In addition to identifying nuclear Survivin as a potential
prognostic marker for prostate cancer, these results strongly suggest that assessment
of Survivin levels in distinct subcellular compartments, rather than overall Survivin
expression, may be critical when correlating Survivin levels to clinical outcome and
clinicopathologic characteristics.
We identified an association between Grp78 and Survivin expression in stage
T
3
N
0
M
0
prostate cancer patients who have not undergone preoperative hormonal
therapy. It has recently been suggested that Survivin may play a role in regulating
response to stress to the endoplasmic reticulum (ER) [Wang 2007]. It is possible that
increased Survivin levels may induce or directly regulate expression of proteins
involved in ER stress response, although further mechanistic studies on Survivin
action during cellular stress are necessary. The current study demonstrates that both
Grp78 and Survivin, anti-apoptotic signals which can bypass AR activation to allow
prostate cancer cell survival under stressful cellular environments, are similarly
upregulated in locally advanced prostate cancer.
Our findings suggest that Survivin expression and cellular distribution may
play important roles in the development of castration resistant prostate cancer.
Nuclear Survivin expression is decreased on initial exposure to androgen ablation
82
therapy in T
3
N
0
M
0
prostate cancer, but is then strongly increased in CRPC. While
both nuclear and cytoplasmic expression of Survivin are associated with tumor
aggressiveness, nuclear Survivin expression appears to be the better marker for
predicting clinical outcome in T
3
N
0
M
0
prostate cancer. Further, we demonstrate that
Survivin and Grp78 are coordinately expressed in prostate cancer. Based on the
current evidence, it is possible that differential cellular localization of Survivin in
prostate cancer cells plays a role in resistance to antiandrogen therapy, and this study
suggests that Survivin may be a rational therapeutic target in the management of
CRPC. Further studies addressing the localization of Survivin and Survivin splice
variants in prostate cancer cells may provide useful mechanistic insight to the role of
Survivin in prostate cancer progression.

83
Chapter Four: Her-2/neu Promotes Prostate Cancer Cell Growth and Survival
and is Involved in Cyclin D1 Regulation

4.1 Introduction
The molecular mechanisms contributing to the development of CRPC are still
not clearly understood. Elucidation of significant molecular alterations that occur
between hormone dependent prostate cancer growth and castration resistant growth
may allow for the identification of which molecular events are responsible for the
development of castration resistance. As state previously, our lab has established a
unique clinical cohort of tumors from prostate cancer patients, designed to represent
the successive stages in the pathway to CRPC. This includes tumors from stage
T
3
N
0
M
0
prostate cancer patients who have not been exposed pre-operatively to
hormone therapy, tumors from stage T
3
N
0
M
0
patients who have been exposed pre-
operatively to hormone therapy, and tumors from patients with castration resistant
prostate cancer. To support and elaborate on observations in this clinical system, we
have also constructed an in vitro system through which the growth environments and
gene expression of cultured cells may be altered so that we may better understand the
effects and pathways associated with our clinical observations.
In contrast to breast cancer, where estrogen insensitivity is often associated
with loss of expression of the estrogen receptor [McGuire 1991], the majority of
castration resistant prostate cancers maintain their androgen receptor expression and
84
androgen dependent gene expression, such as PSA [Visakorpi 1999]. This indicates
that in many cases of CRPC, the androgen growth pathway is still active.
Her2/neu (c-erb B-2) is a proto-oncogene located on chromosome 17q [Ross
1998]. Her-2/neu-encoded p185 is a receptor tyrosine kinase belonging to the family
of epidermal growth factor (EGF) receptors [Ross 1998], and associates with
multiple signal transduction pathways. There is postulated “cross-talk” between the
Her-2/neu and AR pathways through tyrosine and MAP kinase, through which Her-
2/neu can activate the androgen receptor in the absence of androgen and enhance the
magnitude of response of the AR, even when exposed to low levels of androgen
[Craft 1999, Yeh 1999].
We previously identified that receptor tyrosine kinase Her-2/neu is
significantly overexpressed in castration resistant tumors as compared to hormone
dependent tumors, and that expression of Her-2/neu is induced upon androgen
withdrawal treatment in clinical samples [Shi 2001]. We further examined the
molecular mechanisms through which Her-2/neu may contribute to the development
of CRPC using an in vitro system in which a hormone sensitive prostate cancer cell
line, LNCaP, was stably transfected with Her-2/neu cDNA. Utilizing this
established cell line model of castration resistant growth, we found that Her-2/neu
overexpression may promote castration resistant proliferation and survival through
modulation of key regulators in the cell cycle, signal transduction, and apoptosis,
such as p27, Akt, and Bcl-2 [Shi 2006]. It was shown that in prostate cancer cells
that overexpressed Her-2/neu (LNHer), apoptotic index was markedly decreased in
85
these cells as compared to mock-transfected LNCaP cells after prolonged growth in
androgen-deprived medium. The molecular changes that accompanied this
inhibition of apoptosis included upregulation of Bcl-2, suppression of the
upregulation of p27, increase of phosporylated Akt, and augmented PSA expression.
These studies suggest that Her-2/neu overexpression provides an alternative growth
and survival stimulus for prostate cancer in androgen-deprived conditions by: (1)
promoting castration resistant cell proliferation via loss of cell cycle control, (2)
promoting castration resistant prostate cancer cell survival via upregulation of Bcl-2,
and (3) activating the AR in the absence of androgen. Further, these effects may be
mediated, at least in part, by the activation of the Akt pathway.
In addition to growth signaling via signal transduction pathways, Her-2/neu
may have alternate mechanisms through which it stimulates cellular growth and
proliferative processes. One such potential pathway is modulation of signals critical
for maintenance of the cell cycle. The cyclins are a family of cell cycle regulatory
proteins that modulate the activity of the family of serine/threonine kinases, termed
Cdks [Pestell 1999]. Cyclin D1 functions as the rate-limiting regulatory subunit of a
multi-protein cyclin/Cdk complex that is critical for controlling progression through
the G1- to S- phase of the cell cycle [Pestell 1999]. Deregulation of cyclin D1 gene
expression has been described in numerous types of human tumors and the tissue
specific dependency of certain oncogenes on cyclin D1, including Ras, ErbB-2 and
b-catenin [Lee 2000, Yu 2001, Hulit 2004] is well documented.
86
Cyclin D1 mRNA levels were induced by EGF in human prostate cancer cell
lines [Perry 1998] and were found to be increased in both primary prostate cancer
samples [Han 1998] and androgen-independent bone metastases [Drobjnak 2000].
Increased expression of cyclin D1 in LNCaP human prostate cancer cells enhanced
cell growth and tumorigenicity [Perry 1998, Chen 1998] while inhibition of ErbB-
2/ErbB-3 signaling by the flavonoid, quercetin, reduced cyclin D1 protein levels,
resulting in an inhibition of cell cycle progression both in prostate cancer cell lines
[Huynh 2003] and in vivo [Ma 2004]. These data are all consistent with a role for
cyclin D1 as a mediator of prostate epithelial cell proliferation. We were interested
in the role of cyclin D1 as a potential signal through which receptor tyrosine kinase
Her-2/neu modulates prostate cancer cell growth and proliferation.

4.2 Materials and Methods
4.2.1 Patient Population
The recruitment and studies of patients described here have been approved by
local institutional review boards. This study included tumor samples from 36
patients with prostate cancer, who were classified as pathological stage T
3
N
0
M
0

disease [UICC 2002]. Specimens were obtained through radical retropubic
prostatectomy with bilateral pelvic lymph node dissection at the University of
Southern California/Norris Comprehensive Cancer Center between 1982 and 1996.
Patient follow-up was done as described in Chapter 2.2.2.

87
4.2.2 Immunohistochemical Staining (IHC)
Immunohistochemical staining for cyclin D1 was performed on human
prostate cancer specimens which we had previously assessed
immunohistochemically for Her-2/neu [Shi 2001]. IHC was performed as described
in Chapter 2.2.3. Briefly, the slides were blocked with normal horse serum for 20
minutes, and incubated for 1 hour with anti-cyclin D1 mouse monoclonal antibody
(Novacastra Laboratories, Newcastle upon Tyne, United Kingdom) at a 1:20 dilution
in phosphate-buffered saline. Incubation with biotinylated horse anti-rabbit
secondary antibody at a 1:200 dilution was followed by avidin-biotin-conjugate
(ABC, Vector Laboratories, Inc., Burlingame, CA). Chromogen of 0.03%
diaminobenzidine was then applied, with hematoxylin counterstaining. Negative
controls consisting of diluent with no antibody, and positive tonsillar tissue controls
with heterogeneous immunoreactivity were used in all experiments.

4.2.3 Immunoreactivity Assessment of Clinical Samples
All slides examined for cyclin D1 immunoreactivity were interpreted by a
pathologist (MA), who was blinded to all outcome data. Scoring of tumor sections
for cyclin D1 was categorized based on 2 criteria: 1) percent of tumor cells
demonstrating nuclear immunoreactivity, and 2) intensity of nuclear
immunostaining. For assessment according to percent of cells with nuclear
immunoreactivity, tumors were classified as showing weak cyclin D1 expression
(<5%), moderate cyclin D1 expression (5-10%), or strong cyclin D1 expression
88
(>10%). For intensity of nuclear immunoreactivity, tumors were classified as having
weak or no cyclin D1 expression (1+), moderate cyclin D1 expression (2+), or strong
cyclin D1 expression (3+). Cyclin D1 was then categorized as a combined measure
(i.e. status) of percent of immunoreactive tumor cells and intensity of nuclear
immunoreactivity. Cyclin D1 status was assigned as negative to cases with <5%
cyclin D1 immunoreactivity or weak (1+) staining. All other cases were assigned
positive cyclin D1 status. Due to the heterogeneous nature of prostate cancer,
scoring was performed using an overall evaluation of the entire tissue section and by
counting the average percentage of immunoreactive tumor cells per total number of
tumor cells in 5 high power fields (hpf) per slide. Her-2/neu membrane
immunoreactivity was assessed as previously described [Shi 2001]. For statistical
evaluation, Chi-Square analysis was used to compare cyclin D1 expression and Her-
2/neu expression in the clinical specimens. Results were considered significant at
the p<0.05 level for 2-sided analyses.

4.3 Results
Expression levels of cyclin D1 (Figure 4-1) and Her-2/neu were assessed in
36 stage T
3
N
0
M
0
prostate tumors using immunohistochemistry. Of the 36 prostate
tumors immunostained for cyclin D1, 33% (n=12) tumors were found to be positive
for cyclin D1 (combined intensity and percent tumor cells reactive) (Table 4-1).
Her-2/neu expression was considered positive in 9 (25%) of these 36 cases (Table 4-
1). Cyclin D1 expression was strongly associated with Her-2/neu expression,
89
according to cyclin D1 and Her-2/neu status (p<0.0001). Furthermore, when we
examined the correlation between Her-2/neu status and expression of cyclin D1
either by intensity of immunoreactivity or percent of immunoreactive tumor cells,
the expression levels of cyclin D1 and Her-2/neu were also significantly correlated
(p=0.0081 and p=0.0018, respectively). These data demonstrate that increased Her-
2/neu expression correlates with increased cyclin D1 protein levels in clinical
prostate cancer.

90
Figure 4-1. Cyclin D1 expression in prostate cancer. Immunohistochemical
staining was performed on prostate cancer specimens previously screened for ErbB-2
expression. A) Tumor from a T
3
N
0
M
0
patient negative for cyclin D1 nuclear
immunoreactivity. B) T
3
N
0
M
0
tumor showing weak (1+) focal cyclin D1 nuclear
immunoreactivity in >10% of tumor cells. C) T
3
N
0
M
0
tumor showing moderate (2+;
arrow) focal immunoreactivity in 5-10% of tumor cells. D) T
3
N
0
M
0
tumor showing
weak (arrowheads) and strong (arrows) cyclin D1 nuclear immunoreactivity.
Original magnification in all panels was x200.

91
Table 4-1. Correlation between ErbB-2 and Cyclin D1 immunostaining in clinical
specimens

92
4.4 Discussion
Accumulating clinical evidence from our group and others has clearly
demonstrated that increased expression of Her-2/neu contributes to human prostate
disease [Sanchez 2002, Morris 2002, Myers 1994, Signoretti 2000, Shi 2006]. These
findings are further supported by both in vitro [Shi 2006, Mellinghoff 2004, Craft
1999] and in vivo [Mellinghoff 2004, Gregory 2005] studies. Previous data had also
established that the activity of cell cycle regulatory protein, cyclin D1, is important
for both cellular proliferation and regulation of AR function [Reutens 2001, Lim
2005]. We assessed the possibility that cyclin D1 may play a role in Her-2/neu
signaling in prostate cancer cells. We showed that immunostaining for Her-2/neu
and cyclin D1 in clinical specimens revealed a statistically significant correlation
between Her-2/neu and cyclin D1 positivity. Additional data from our group also
demonstrated that overexpression of either wild-type or constitutively activated Her-
2/neu induced cell cycle progression, cyclin D1 promoter, and cyclin D1 gene
activity in prostate cancer cell lines (data not shown) [Casimiro 2007]. This study,
combined with the in vitro and in vivo studies demonstrating Her-2/neu regulation of
cyclin D1 in prostate cancer cells [Casimiro 2007], suggests that upregulation of
cyclin D1 may be an additional mechanism through which overexpression of Her-
2/neu may contribute to prostate cancer cell proliferation, and provides additional
evidence that Her-2/neu may serve as an effective therapeutic target for the
management of prostate cancer.

93
Chapter Five: Targeting Molecular Mechanisms Involved in Cell Growth,
Proliferation and Survival: Effect on Castration Resistant Cells

5.1 Introduction
Emphasis has been placed on the idea that combinatorial therapies that inhibit
multiple specific gene targets can produce synergistic effects and provide the basis
for identifying additional anti-apoptotic genes that may serve as targets in a multi-
agent approach to enhance the activity of existing therapies [Gleave 2005]. For
example, markers of different gene families have been shown to directly interact
with the same caspases to inhibit apoptosis [Reddy 2003, Shin 2001]. Thus,
abrogating the activity of one anti-apoptotic signaling molecule clearly still allows
for other mechanisms through which cancer cells can escape apoptosis. The
importance of studies such as this, in which multiple anti-apoptotic targets are
examined, is identifying which prospective targets of inhibition, or combination of
targets, are most essential for cancer cell survival under particular stress conditions,
such as prostate cancer cells surviving androgen deprivation. Testing of inhibition of
these key molecules should provide insight as to which therapies would be most
effective in impeding persistent cancer cell survival under current available
therapies. Treatment with siRNA allows for targeted inhibition of transcription.
Several studies have reported that siRNA inhibition of Grp78 or Survivin siRNA
treatment stimulates cancer cell apoptosis, inhibits cell growth, and sensitizes tumor
cells to other anticancer therapies [Dong 2005, Tsutsumi 2005, Wang 2005, Paduano
94
2006, Yonesaka 2005]. We were interested in examining the individual and
combined effects of these molecular antagonists, along with inhibitors of growth
signals, on prostate cancer cell survival.

5.2 Materials and Methods
5.2.1 Cell Culture
C42B cells were grown in RPMI 1640 medium (Invitrogen, Carlsbad, CA)
with 50units/mL penicillin, 50 units/mL streptomycin, and 10% fetal calf serum
(FCS) (Mediatech, Inc. Herndon, VA). All cells were maintained in a humidified
incubator at 5%CO
2
and 37°C.

5.2.2 siRNA Transfection and Herceptin Treatment
Castration resistant C42B prostate cancer cells were transfected with siRNA
against Grp78 with the sequence 5’-AAGGUUACCCAUGCAGUUGUU-3’ (Dong
2005), and/or with siRNA against Survivin with the sequence 5’-UGUAGAGAUG-
CGGUGGUCC-3’ [Paduano 2006], or with control siRNA with the sequence 5’-
AAGGUGGUUGUUUUGUUCACU-3’ which does not match to any human
sequence [Dong 2005]. All siRNA’s were purified duplexes with two extra
thymidine bases, forming 3’ overhangs on both strands. Twenty-four hours before
transfection, C42B cells were seeded at a density of 5.0 x 10
5
cells per 25-cm
2
flask.
Each siRNA was incubated with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and
Opti-MEM medium (Invitrogen, Carlsbad, CA) for 20 minutes, according to
95
manufacturer’s protocol. Final mixtures were then added to each flask to give a final
concentration of 100nmol/L for each siRNA. Cells were incubated with siRNA for 4
hours at 37˚C, then replaced with RPMI 1640 medium containing 10% fetal calf
serum. For inhibition of Her-2/neu activity, Herceptin monoclonal antibody for Her-
2/neu (Genentech, South San Francisco, CA) was added to C42B cells at a final
concentration of 20 μg/mL. Further analysis was performed following incubation for
24 hours at 37˚C.

5.2.3 Western Blot
For western blot analysis, cell lysates from C42B cells were prepared by
lysing in 1mL ice-cold RIPA buffer. Equal amounts of total protein from each
sample were subjected to SDS-PAGE in a 4-20% Tris-HCl gel (NuSep, Sydney,
Australia). Following electrophoresis, the proteins were transferred to a pure
nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). The membrane was
then incubated in Odyssey Blocking Buffer (Li-Cor Biosciences, Lincoln, Nebraska),
followed by overnight incubation with primary rabbit polyclonal anti-Grp78
antibody (1:200 dilution; Santa Cruz Biotechnology Inc, Santa Cruz, CA),
monoclonal Survivin antibody (1:500 dilution; Santa Cruz Biotechnology, Santa
Cruz, CA), rabbit polyclonal Her-2/neu antibody (1:600; Dako, Glostrup, Denmark),
or rabbit polyclonal Apaf-1 antibody (1: 500; LabVision, Fremont, CA). Signal
detection was performed using Alexa Fluor 680 goat anti-rabbit antibody (Molecular
Probes, Eugene, Oregon) or IRDye 800 rabbit anti-mouse antibody (Rockland
96
Immunochemicals, Gilbertsville, PA) and subsequent scanning of the membrane by
the Odyssey Infrared Imager (Model 9120, Li-Cor Biosciences, Lincoln, Nebraska).
All bands from western analysis were quantified for protein expression with Odyssey
Infrared Imaging Software (Li-Cor Biosciences, Lincoln, Nebraska) to assess
integrated intensity (pixel volume) as a measure of optical density. Band density
was represented as the ratio of average band intensity (I) of each sample to the
average band intensity of the corresponding β actin control band. Experiments were
performed in triplicate.

5.2.4 Annexin Binding Assay for Early Apoptosis
For assessment of early-stage apoptosis, C42B cells were analyzed for
externalization of phosphatidylserine (PS) on the plasma membrane using FITC-
conjugated Annexin V. Assay was performed according to manufacturer’s protocol
(Invitrogen, Carlsbad, CA). Briefly, following siRNA transfection, C42B cells were
washed with cold PBS, then incubated at room temperature for 15 minutes with
annexin V and propidium iodide (PI). Fluorescence was detected and analyzed using
Image-Pro 5.1 Software (Media Cybernetics, Bethesda, MD). The level of apoptosis
per sample was expressed as apoptotic index, calculated as the number of annexin-V-
positive cells divided by total number of tumor cells per high power field (hpf) for
1000 cells. Cells that were PI-positive were considered indistinguishable from
necrotic cells and were excluded.

97
5.3 Results
5.3.1 Optimization of siRNA Interference
To assess optimal conditions for siRNA transfection, castration resistant
C42B cells were incubated with Grp78 siRNA or Survivin siRNA for 24, 48, or 96
hours at concentrations of 10 nM, 50 nM, 100 nM, 125 nM, or 150 nM of siRNA in
growth media (Figure 5-1). Using western blot analysis, we found that both Grp78
siRNA transfection and Survivin siRNA transfection in C42B cells resulted in
knockdown of protein expression as compared to control siRNA (Figure 5-1).
Optimal incubation time with Grp78 siRNA for suppression of Grp78 protein
expression was 24 hours (Figure 5-1A). Similarly, highest levels of Survivin protein
expression inhibition were seen after 24 hours of incubation with Survivin siRNA
(Figure 5-1C). Concentrations of siRNA demonstrating greatest protein knockdown
were 100 nM for both Grp78 siRNA and Survivin siRNA transfection. Comparison
of band pixel intensity ratios (intensity of sample band divided by intensity of beta
actin band) showed that inhibition of Grp78 protein expression was 85% greater in
C42B cells which underwent Grp78 transfection with 100 nM Grp78 siRNA
followed by 24 hour incubation, compared to that in C42B cells treated with control
siRNA under the same conditions. Knockdown of Survivin expression by 100 nM of
Survivin siRNA followed by 24 hour incubation was found to be 80% greater than in
C42B cells treated with control siRNA under similar conditions. Increased
concentrations of siRNA beyond 100 nM did not result in greater suppression of
protein expression for Grp78 or Survivin (Figure 5-1B, D).
98
5.3.2 Relative Effects of Non-specific siRNA on Induction of Apoptosis
Because we employed varying amounts of non-specific reagents which may
induce apoptosis in cultured cells, we evaluated the apoptotic effects of increasing
the concentration of these reagents in cultured prostate cancer cells. Using western
blot analysis, we assessed the relative levels of Apaf-1 following treatment of
castration resistant C42B prostate cancer cells with Lipofectamine 2000 transfection
reagent only and non-specific control siRNA with Lipofectamine 2000 in single (1%
lipid solution, 100 nM siRNA) and double (2% lipid solution, 200 nM siRNA)
concentrations. According to the band pixel intensity ratio, measured as intensity of
the Apaf-1 band divided by the intensity of the corresponding beta actin band, we
found that expression of Apaf-1 following treatment with transfection reagent and/or
control siRNA was not significantly different when amounts were doubled, although
Apaf-1 expression was slightly increased upon treatment with 200 nM control
siRNA (Figure 5-2).

5.3.3 Induction of Apoptosis by Combined Targeting in Castration Resistant
Prostate Cancer Cells

5.3.3.1 In Vitro Analysis of Apaf-1 Expression Following siRNA and Herceptin
Treatment in Castration Resistant Cells
We performed single and combination treatments of Herceptin, Grp78
siRNA, and Survivin siRNA on castration resistant C42B prostate cancer cells and
99
evaluated relative protein expression of Apaf-1, Grp78, Survivin, and Her/2/neu in
cell lysates obtained from harvested cells which underwent varying treatments.
Western blot analysis for Her-2/neu, Grp78, and Survivin, confirmed appropriate
protein knockdown during different treatments, compared to negative control siRNA
and lipofectamine only treatments (Figure 5-3A). Assessment of apoptosis induction
using band pixel intensity ratios (ratios of band intensity of Apaf-1 divided by band
intensity of corresponding beta actin) demonstrated that apoptosis occurred in all
samples. Apaf-1 expression was shown to be lowest in control siRNA,
lipofectamine, and Herceptin treated cells (average band intensity ratios of 9.4, 9.7,
and 3, respectively), greater in single target siRNA-treated cells incubated with
Grp78 siRNA or Survivin siRNA (ratios of 10.5 and 11.5), even greater in double
target siRNA and/or Herceptin-treated cells incubated with Herceptin and Grp78
siRNA, Herceptin and Survivin siRNA, or Grp78 siRNA and Survivin siRNA (ratios
of 19.1, 20.7, and 18.5, respectively), and highest in C42B cells treated with
Herceptin, Grp78 siRNA, and Survivin siRNA in combination ( average ratio of
30.6), as shown in Figure 5-3B.

5.3.3.2 Evaluation of Apoptotic Index During Early-Stage Apoptosis
Single target and combination target-treated castration resistant C42B cells
were examined for early apoptosis, following incubation with FITC-conjugated
annexin V and propidiun iodide (PI). All samples showed adherent C42B cells
staining positive for annexin V binding (Figure 5-4A). Assessment of apoptosis
100
induction was done using apoptotic indices (AI), where the level of apoptosis per
sample was expressed as apoptotic index, calculated as the number of annexin-V-
positive cells divided by total number of tumor cells per high power field (hpf) for
1000 cells. Similar to protein expression analysis, AI was found to be lowest in
control siRNA, lipofectamine, and Herceptin treated cells (average AI’s of 7.4, 4.6,
and 4.3, respectively), greater in single target and double target-treated cells
incubated with Grp78 siRNA, Survivin siRNA, Herceptin and Grp78 siRNA,
Herceptin and Survivin siRNA, or Grp78 siRNA and Survivin siRNA (average AI’s
of 9.3, 12.8, 11, 19, and 18.5, respectively), and highest in C42B cells treated with
Herceptin, Grp78 siRNA, and Survivin siRNA in combination (average AI of 33.7),
as demonstrated in Figure 5-4B. Because cells that were found to be positive for
both PI and annexin V could not be distinguished as late apoptotic cells or necrotic
cells, these cells were excluded from final analysis of AI.

101
Figure 5-1. Serial dilution of Grp78 and Survivin siRNA transfection. A)
Western blot analysis of Grp78 expression in castration resistant C42B cells
incubated for 24, 48, or 96 hours following 4 hour incubation with siRNA against
Grp78 at 10 nM, 50 nM, or 100 nM, or incubation with non-specific negative control
siRNA at 50 nM or 100 nM B) Western blot analysis of Grp78 expression in C42B
cells incubated for 24 hours following 4 hour incubation with siRNA against Grp78
at 100 nM, 125 nM, or 150 nM, or incubation with non-specific negative control
siRNA at 150 nM C) Western blot analysis of Survivin expression in C42B cells
incubated for 24 hours following 4 hour incubation with siRNA against Survivin at
10 nM, 50 nM, or 100 nM, or incubation with non-specific negative control siRNA
at 50 nM or 100 nM D) Western blot analysis of Survivin expression in C42B cells
incubated for 24 hours following 4 hour incubation with siRNA against Survivin at
100 nM, 125 nM, or 150 nM, or incubation with non-specific negative control
siRNA at 150 nM; beta actin loading control shown in lower panel.

102

A
103

B
104

C
105

D
106

Figure 5-2. Comparison of effects of control siRNA and lipofectamine levels on
apoptosis. Western blot analysis of Apaf-1 in lysates from C42B prostate cancer
cells treated with transfection reagent only at 1% (Lipofectamine 1) and 2%
(Lipofectamine 2) solutions, 100 nM of non-specific control siRNA (Control siRNA
x 1), and 200 nM of non-specific control siRNA (Control siRNA x 2); beta actin
loading control shown in lower panel. Numbers represent the ratio of Apaf-1 band
density to β actin band density, using the lowest ratio as the reference point of 1.00.

107

Figure 5-3. Western blot analysis of induction of apoptosis following combined
targeted treatment in castration resistant C42B cells. C42B prostate cancer cells
were incubated with a single treatment of Herceptin (H), Grp78 siRNA (G), Survivin
siRNA (S), negative control siRNA (C), or lipofectamine only (L), or combination
therapies of Herceptin and Grp78 siRNA (HG), Herceptin and Survivin siRNA (S),
Grp78 siRNA and Survivin siRNA (GS), or Herceptin, Grp78 siRNA, and Survivin
siRNA (HGS) A) Western blot for Her-2/neu, Apaf-1, Grp78, and Survivin,
demonstrating relative protein knockdown during different treatments and induction
of apoptosis. The same cell lysates were run on two separate gels to preserve
analysis of protein expression; both beta actin loading controls are shown below B)
Comparative analysis of apoptosis induction using band pixel intensity ratios (y-
axis); ratios of band intensity of Apaf-1 divided by band intensity of corresponding
beta actin were used to evaluate apoptosis levels during varying treatments (x-axis)
in C42B cells; experiments were done in triplicate, with error bars signifying ranges
of intensity ratios per treatment.

108

A

109
B

110
Figure 5-4. Fluorescence analysis of apoptosis induction through membrane
expression of annexin V. C42B prostate cancer cells were incubated with a single
treatment of Herceptin (H), Grp78 siRNA (G), Survivin siRNA (S), negative control
siRNA (C), or lipofectamine only (L), or combination therapies of Herceptin and
Grp78 siRNA (HG), Herceptin and Survivin siRNA (S), Grp78 siRNA and Survivin
siRNA (GS), or Herceptin, Grp78 siRNA, and Survivin siRNA (HGS) A) Images of
treated castration resistant C42B cells following incubation with FITC-conjugated
annexin V and PI; top panel shows total number of adherent C42B cells (original
magnification x400); middle panel shows annexin V-positive cells (green); lower
panel shows PI-positive cells (red) B) Analysis of apoptosis induction after
treatment (y-axis) in C42B cells using apoptotic indices (AI, x-axis) where the level
of apoptosis per sample was expressed as apoptotic index, calculated as the number
of annexin-V-positive cells divided by total number of tumor cells per high power
field (hpf) for 1000 cells; experiments were done in triplicate, with error bars
signifying ranges of apoptotic indices per treatment C) Comparison of quantitation
of induction of apoptosis using Apaf-1 expression and apoptotic index (AI); AI
levels were normalized to Apaf-1 expression as measured by western blot.

111

A

H G S HG HS GS L C HGS
Total
Annexin+
PI+
112

B

113
C

114
5.4 Discussion
The current challenge in treatment of advanced prostate cancer is that patients
who are initially responsive to androgen ablation therapy often develop CRPC,
despite recent advances in cancer therapy. A better understanding of the complex
contributing molecular mechanisms underlying the development of CRPC is
necessary so that the most appropriate therapeutic targets may be identified.
We have previously established an in vitro cell line models which allows for
molecular assessment of signal transduction events in the progression to CRPC, and
permits testing of novel rational therapeutics. Using this model, we identified that
overexpression of receptor tyrosine kinase Her-2/neu plays a significant role in
castration resistant cell proliferation and survival [Shi 2006]. In recent years,
involvement of many additional proteins in the development of castration resistance
has been suggested by our group and others, including induction of survival-
associated proteins Survivin and Grp78 [Mintz 2003, Zhang 2005, Pootrakul 2006].
Survivin, a nuclear and cytosolic protein with antiapoptotic function, has been shown
to promote resistance to antiandrogen treatment in prostate cancer, both in vitro and
in vivo [Fortugno 2002, Zhang 2005]. We also found that Survivin expression and
cellular localization plays a role in CRPC development. Grp78 functions as an
endoplasmic reticulum (ER) ‘chaperone’ protein, and induction of Grp78 is known
to be upregulated in stress conditions often seen in the tumor microenvironment [Lee
2001]. Thus, we applied our in vitro model using castration resistant C42B cells, a
cell line derived from LNCaP cells, to investigate the effects of combinatorial
115
targeting of key signals in cell survival and proliferation on castration resistant
growth.
Using two different experimental systems for analysis of apoptosis,
evaluation of apoptotic signal Apaf-1 protein expression and assessment of PS
externalization in early apoptosis, we found that targeting of Grp78 or Survivin alone
resulted in greater apoptosis induction than inhibition of Her-2/neu activity with
Herceptin treatment. While this may be due, in part, to the toxicity of the
lipofectamine transfection reagent for siRNA, it corroborates the evidence that the
major role for Her-2/neu in prostate cancer cells is in activation of alternate growth
pathways, where the inherent functions of Grp78 and Survivin are anti-apoptotic.
Thus, inhibition of Grp78 or Survivin alone may have more adverse effects on cell
survival than suppression of Her-2/neu activity alone. We also found that the double
target combination which appeared to be most effective in inducing apoptosis in
castration resistant prostate cancer cells was inhibition of Her-2/neu and Survivin.
While inhibition of Grp78 would be detrimental to cellular response to
environmental stress, knockdown of Survivin expression may affect a wider range of
normal cellular functions, including regulation of apoptosis and normal mitosis, and
suppression of Her-2/neu activity would also inhibit the functions of several
downstream signals of the Her-2/neu pathway. Thus it is plausible that inhibition of
these two biomolecular signals in combination would result in greater apoptosis
induction than other double combinations.
116
The synergistic affect on apoptosis induced by inhibiting the activity of Her-
2/neu, Grp78, and Survivin in combination was also demonstrated by both Apaf-1
expression and annexin V binding analysis. Complexities arising from the
heterogeneity of prostate tumors and adaptability of tumor cells necessitate the use of
therapies with multiple targets [Gleave 2005]. Our findings suggest that there may
be critical pathways or networks in castration resistant cells that if suppressed can
lead to optimal induction of cell death, where the crucial step is in identifying the
most effective combination of targets. Future studies require further identification of
key rational multitarget therapies that will also balance practicality and feasibility of
treatment during the management of advanced prostate cancer.

117
Chapter Six: Conclusions and Impact
The current treatment for advanced prostate cancer is hormonal therapy.
Despite these treatments, progression to castration resistant disease occurs in the
majority of these patients, and thus remains a major obstacle in the management of
prostate cancer. Although it is widely known that the androgen receptor (AR) is
expressed and remains active in CRPC, it has been suggested that molecular
mechanisms bypassing AR activation may be an alternative pathways through which
prostate cancer cells continue to survive and grow despite androgen depletion
[Feldman 2001].
To further assess these potential AR bypass signals in the development of
castration resistance, we established two experimental models to represent the
development of CRPC. The first is a clinical model using archived prostate tumors
from 3 prostate cancer patient groups, including stage T
3
N
0
M
0
patients who were not
exposed preoperatively to androgen ablation therapy, stage T
3
N
0
M
0
patients who
have been exposed to androgen ablation treatment, and CRPC patients. The second
model is an in vitro system including cultured hormone dependent LNCaP prostate
cancer cells, grown in androgen enriched or androgen depleted medium, and
castration resistant C42B cells, derived from LNCaP cells.
Applying our models of the development of castration resistant disease, we
first examined the role Grp78, an ER stress response protein and molecular
chaperone, in CRPC development. Because the transition from androgen dependent
to castration resistant prostate cancer requires the survival of tumor cells under the
118
stressful condition of androgen depletion, we hypothesized that Grp78 may confer
protection to prostate cancer cells and would thus be upregulated. We found that
Grp78 was highly expressed in CRPC as compared to localized prostate cancer, and
induction of Grp78 expression upon androgen withdrawal was confirmed in our cell
line model [Pootrakul 2006].
Our studies have also demonstrated the importance of inhibitor of apoptosis
Survivin in the development of CRPC, where Survivin nuclear expression becomes
depleted upon androgen withdrawal and Survivin is subsequently strongly expressed
in both the nucleus and cytoplasm in CRPC tumors, compared to localized prostate
cancer. These findings were also corroborated by in vitro experiments on both
whole cell extractions and compartmentalized extractions of the nucleus and
cytoplasm.
Evidence from our group has shown that receptor tyrosine kinase Her-2/neu
is induced upon androgen ablation therapy in hormone dependent tumors and is
expressed highest in CRPC tumors [Shi 2001]. We have also found that Her-2/neu
overexpression results in downstream molecular alterations that can contribute to
prostate cancer cell proliferation and survival, including upregulation of Bcl-2,
suppression of p27, and Akt activation [Shi 2006]. In this study, we found that the
expression of Her-2/neu and cell cycle regulator cyclin D1 are significantly
associated in stage T
3
N
0
M
0
prostate tumors, suggesting another potential mechanism
through which Her-2/neu may contribute to cell proliferation, and providing further
evidence that Her-2/neu may be a rational target for prostate cancer treatment.
119
Figure 6-1. Potential therapeutic targets

In this study, we have identified two anti-apoptotic signals that may
contribute to the development of castration resistance. Grp78 is induced upon
androgen withdrawal in hormone dependent prostate cancer cells, while overall
Survivin expression is suppressed in hormone dependent cells upon androgen-
depletion, where the majority of Survivin decreased expression occurs in the nuclear
compartment of prostate cancer cells. Both Grp78 and Survivin are then highly
upregulated in CRPC tumors, suggesting that these signals play important roles in
120
castration resistant growth. Along with previous and current data from our group,
demonstrating the significance of Her-2/neu in the development of CRPC, and the
numerous pathways through which Her-2/neu may act to promote cell growth,
proliferation, and survival, these studies have identified Grp78, Survivin, and Her-
2/neu as potential candidates for targeted therapy.

Figure 6-2. Targeting basic hallmarks of cancer

*Adapted from Hanahan and Weinberg 2000

We found that targeting these three signals in combination results in markedly
increased apoptosis in castration resistant prostate cancer cells, as compared to
singular or paired suppression of the three molecular targets. Because there are
121
several additional signals thought to be involved in the development of CRPC, our
future goals are to identify and evaluate the best possible combination of targets that
will safely and effectively provide maximum induction of apoptosis in castration
resistant cells, for appropriate use in the clinical setting and optimal management of
advanced prostate cancer.

122
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Abstract (if available)

Abstract A significant clinical problem is that patients who are initially responsive to androgen ablation therapy often develop castration resistant carcinoma of the prostate (CRPC), despite recent advances in cancer therapy. A better understanding of the complex contributing molecular mechanisms underlying the development of CRPC is necessary so that the most appropriate therapeutic targets may be identified. In order to further our understanding of the development of castration resistance, we have established a unique clinical cohort designed to represent successive stages in the development of CRPC. Immunohistochemical analysis (IHC) on clinical specimens allows for identification of potential prognostic markers of prostate cancer, and the use of in vitro cell line models allows for molecular manipulation and assessment of signal transduction events in the progression to CRPC, and will also permit testing of novel rational therapeutics. Our lab has previously identified that overexpression of receptor tyrosine kinase Her-2/neu plays a significant role in castration resistant cell proliferation and survival [Shi 2006]. Recently, involvement of many additional proteins in the development of castration resistance has been suggested, including induction of survival-associated proteins Survivin and Grp78 (78 kDa glucose-regulated protein). Survivin has been shown to promote resistance to antiandrogen treatment in prostate cancer, both in vitro and in vivo [Fortugno 2002, Zhang 2005]. Immunoreactivity of stress response protein Grp78 has been identified as a putative marker of CRPC [Mintz 2003], while preclinical data has validated Grp78 as a functional cell surface molecular target for prostate cancer [Arap 2004].

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Pootrakul, Llana (author)

Core Title Targeting molecular signals involved in the development of castration resistant prostate cancer

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Pathobiology

Publication Date 11/05/2007

Defense Date 07/31/2007

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag OAI-PMH Harvest,prostate cancer

Language English

Advisor Cote, Richard J. (committee chair), Coetzee, Gerhard A. (committee member), Datar, Ram H. (committee member), Dubeau, Louis (committee member)

Creator Email lpootrakul@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m906

Unique identifier UC1462769

Identifier etd-Pootrakul-20071105 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-588404 (legacy record id),usctheses-m906 (legacy record id)

Legacy Identifier etd-Pootrakul-20071105.pdf

Dmrecord 588404

Document Type Dissertation

Rights Pootrakul, Llana

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu