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Exploration of the roles of cancer stem cells and survivin in the pathogenesis and progression of prostate cancer

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Exploration of the roles of cancer stem cells and survivin in the pathogenesis and progression of prostate cancer

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EXPLORATION OF THE ROLES OF CANCER STEM CELLS AND SURVIVIN IN THE
PATHOGENESIS AND PROGRESSION OF PROSTATE CANCER

by

Helty Aprilia Adisetiyo

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETIC, MOLECULAR, AND CELLULAR BIOLOGY)

December 2012

Copyright 2012 Helty Aprilia Adisetiyo

ii

“If we succeed in giving the love of learning, the learning itself is sure to
follow.”
John Lubbock, English biologist and politician 1834-1913

iii

DEDICATION
I dedicate this dissertation to my amazing family, for their constant love and support
through every phase of my life. To my mother, Tina Viriyanti Adiwidya,
my father, Sandjaya Trikadibusana, my sister, Vitria Adisetiyo,
and my husband, Preston Tamerlane Mielke:
Thank you for being my rock.

iv

ACKNOWLEDGMENTS
First and foremost, I would like to thank my mentor, Dr. Pradip Roy-Burman, for
his constant guidance, support, and patience throughout my graduate study. He acted
like a father to me in lab. He gave me the freedom to think independently and
opportunities and room to grow as a scientist, and provided solutions whenever I hit a
dead end in my projects. I learned many things from Dr. Roy-Burman that shaped me
to be the scientist that I am today, including how to approach science, how to think
critically, and how to present science beautifully in a written form. I admire his many
qualities—his intelligence, passion for science, great attention to detail, and amazing
ability to turn science writing into poetry. He truly cares about all his students and post-
docs and takes great interest in our next career steps. I wouldn’t have made it through
this Ph.D program without his continual encouragement and support.
Second, I am grateful for my thesis committee members, Dr. Baruch Frenkel and
Dr. Young-Kwon Hong, as well as my qualifying exam committee members, Dr.
Elizabeth Lawlor, Dr. Louis Dubeau, and Dr. Randy Widelitz, for giving me insightful and
realistic suggestions and advice on the directions of my projects over the years.
Third, I am also thankful for all my current and previous lab members, both for
their technical assistance and moral support when things got tough during my graduate
studies: Kumkum Mittra, Mengmeng Liang, Lauren Geary, Dr. Linda Pham, Dr. Ari
Aycock-Williams, Dr. Shangxin Yang, Dr. Chun-Peng Liao Dr. Minyoung Lim, Gohar
Saribekyan, , Erik Haw, and Engracia. I would like to thank Dr. Chun-Peng Liao and Dr.
Shangxin Yang for taking me under their wings when I first started in the laboratory.
They were great and inspiring mentors to me, from whom I learned a lot of the current

v

experimental methods that I know today. I am grateful for their technical expertise and
troubleshooting suggestions, as well as helpful discussions about my projects and
advice on my career goals. I would like to thank Dr. Shangxin Yang and Dr. Minyoung
Lim for their earlier work that paved the way for my current survivin project, as well as
Dr. Chun-Peng Liao for first involving me in the cancer stem cell work. Gohar
Saribekyan and Erik Haw helped tremendously with the breeding and maintenance of
our mouse colonies and Gohar also trained me on some of the mouse work. I am so
blessed to have Kumkum Mittra, who is like my lab mom, for all her efficient hard work
in managing the lab and making sure we get all our supplies on time, and for the
countless times she has made me laugh, listened and offered her wise advice on
various matters, ranging from scientific to personal. I am so grateful for Mengmeng
Liang, whom I call my twin, for being such an amazing labmate and friend, who
brightened my days with her colorful stories and who was always there for me when I
needed her, both with my projects and personal life.
Fourth, I am indebted to Dr. Chun-Peng Liao, Mengmeng Liang, Dr. Joseph
Jeong (in vivo renal capsule assay), and Lora Barsky (flow cytometry) for their
contribution to the cancer stem cell project. I am grateful to many people for my survivin
project: Mengmeng Liang for her technical assistance, especially in
immunohistochemistry, Dr. Ari Aycock-Williams for her help on tissue sectioning and
H&E staining, Dr. Chun-Peng Liao and Dr. Michael B. Cohen of University of Utah for
their histological analysis, Dr. Edward Conway of University of British Columbia for
contributing the survivin floxed mice, Shili Xu for western blot analysis assistance, Dr.
Nouri Neamati for letting his student Shili help me with my project, Chieh-Yang Cheng

vi

of Cornell University for his help in sectioning some of my paraffin-embedded tissues
and performing H&E staining as well as providing protocols and advice on H&E and
immunohistochemistry, and most importantly, Dr. Alexander Yu Nikitin of Cornell
University for his extensive and thorough critical analyses of my numerous mouse
prostate tissue sections that led to essential results that the paper will be based on and
lending his student Chieh-Yang’s help to me. I would also like to thank Dr. Epstein’s
lab, Dr. Florence Hoffman, Dr. Man-Chung Ting of Dr. Maxson’s lab, Dr. Emily Zamalea
of eBioscience, Niyati Jhaveri of Dr. Hoffman’s lab, Dr. Vivian Medina of Dr. Stiles’ lab,
Ying Liu and Christine Marion of Dr. Dubeau’s lab for helpful technical discussions and
for sharing reagents and lab equipments.
Fifth, I would like to acknowledge my good friends with whom I started the PIBBS
program: Dr. Hyunjung Kim, Dr. Bernice Aguilar, Dr. Vivian Medina, Dr. Pao-Chen Li,
and Dr. Jennifer-Ann Bayan. They were my support group—we cried and laughed
together while sharing our deepest personal heartaches and greatest joys, and I
wouldn’t have made it through the program without them. I would also like to thank my
other scientist friends: Dr. Daniel Boutz, Dr. Morgan Beeby, Dr. Natasha Emmerson,
Anna Skylar, Dr. Shiho Tanaka, Dr. Shila Mekhoubad, Dr. Shilpa Sambashivan, Dr.
Michael Sawaya, Dr. Vanessa Yu, and Mai Phan for their support and extremely helpful
advice during the course of my graduate studies.
Sixth, I am also grateful to Dr. Debbie Johnson, Dr. Ite Laird-Offringa, Marisela
Zuniga, Dawn Burke, Raquel Gallardo, Lisa Doumak, John Johnson, and Raquel
Rodriguez for their tremendous support in many administrative matters pertaining to the
graduate program: rotation, enrollment in classes, qualifying exams, stipend checks,

vii

registration in annual departmental retreats, and training grant processing. I am so
thankful to the California Institute of Regenerative Medicine for granting me the pre-
doctoral training award that helped fund my cancer stem cell project.
Last but not least, I am indebted to my family: mom, Tina Viriyanti Adiwidya, dad,
Sandjaya Trikadibusana, sister, Vitria Adisetiyo, and husband, Preston Tamerlane
Mielke. I would be nothing without them in my life. I thank my mother from the bottom
of my heart for her endless self-sacrifice for her family. She gave up her career in
medicine in Indonesia and moved to the United States with my father, where her
children can enjoy enormous opportunities to thrive. I admire her incredible strength in
the midst of adversity, independence, work-ethics, practical sense, and her many
talents on top of her intelligence. She is an amazing woman, a role model who I always
look up to and I wouldn’t be where I am today if it wasn’t for her. I greatly appreciate my
dad for being such an inspiration to me in his thirst for knowledge, love of science, high
educational aspirations, and quest to use his training in science and medicine to help
others. I am so blessed to have my sister, who is also my best girlfriend, to whom I can
confide to about anything. She’s an excellent graduate student, who is hardworking,
meticulous, efficient, and extremely organized, and has given me incredible support and
advice as we went through Ph.D programs together. I am extremely thankful for my
husband, who I am lucky to call my best guy friend. He takes good care of me like no
man ever has, supports and comforts me in my low times, watches out for my best
interest, and makes me laugh every day that I spend with him. Most of all, I am forever
grateful to God, who has blessed me so abundantly with so many opportunities and
incredible people in my life.

viii

TABLE OF CONTENTS
Epigraph ii
Dedication iii
Acknowledgments iv
List of Tables x
List of Figures xi
Abstract xii
Chapter One: Introduction
1.1 Prostate cancer 1
1.2 Cancer stem cells 2
1.3 Pten deletion mouse model of prostate cancer 3
1.4 Survivin 5
1.5 Hypotheses and experimental strategies 8
References 12

Chapter Two: Study of Cancer Stem Cells and Cancer-associated
Fibroblasts from Tumors of a Mouse Model of Prostate Cancer
2.1 Abstract 15
2.2 Introduction 16
2.3 Materials and methods 18
2.4 Results 21
2.5 Discussion 41
References 47

Chapter Three: Role of Survivin in Prostate Tumorigenesis
3.1 Abstract 51
3.2 Introduction 52
3.3 Materials and methods 54
3.4 Results 59
3.5 Discussion 79
References 85

ix

Chapter Four: Conclusions and Remarks About Future Studies
4.1 Conclusions 90
4.2 Future studies 92
References 95

Bibiliography 96

Appendix: Abbreviations 105

x

LIST OF TABLES

Table 2-1: Detection of prostatic glandular structures in grafts 32

Table 2-2: Grafts containing glandular structure(s) 39

Table 3-1: Prostate pathology of cPten
-/-
mice with monoallelic or
biallelic deletion of survivin 68

Table 3-2: Prostate pathology observed in the cPten
-/-
S
+/-

experimental group 69

Table 3-3: Details of prostate pathology observed in the
cPten
-/-
S
-/-
group 70
Table 3-4: Prostate pathology observed in the cPten
-/-
S
+/+
control
tumor group 71
Table 3-5: Description of histologic evaluation of the prostate in
the Pten
f/f
S
f/f
control group 72

xi

LIST OF FIGURES

Figure 2-1: Analyses of spheroids formed from putative normal and
cancer stem

cells 23

Figure 2-2: Further characterization of spheroids formed 24

Figure 2-3: Spheroid formation analysis with the LSC cell
subpopulations 27

Figure 2-4: Analysis of the T-LSC
hi
cell subpopulation 28

Figure 2-5: In vivo tissue regeneration analysis with tumor-derived cell
subpopulations 31

Figure 2-6: Further characterization of the glandular structures formed
in renal grafts 33

Figure 2-7: Analyses of gene expression in AD-Ca LSC
hi
cells 35

Figure 2-8: In vitro spheroid forming analysis of CRPC LSC
hi
cells 38

Figure 2-9: In vivo renal capsule transplantation assay of AD-Ca and
CRPC LSC
hi
cells 39

Figure 3-1: Survivin deletion has no effect on normal prostate
development 60

Figure 3-2: Breeding scheme for prostate-specific Survivin
deleted mice 62

Figure 3-3: Loss of Survivin in conditional Pten deletion mouse model
delays prostate tumor progression 64

Figure 3-4: Expression pattern of cellular markers in prostates of
conditional Pten knock-out mice with heterozygous and
homozygous deletion of Survivin 75

Figure 3-5: Effects of Survivin deletion on other IAP family
members, apoptosis, and senescence 76

xii

ABSTRACT

Our study of prostate cancer is centered on a conditional mouse model based on
the Cre/lox recombination technology to inactivate the tumor suppressor gene Pten,
whose function is frequently lost in human prostate cancer. This model proved to be a
powerful tool in our investigation on the presence and characteristics of putative cancer
stem cell population and its interaction with the tumor microenvironment, as well as on
the role of survivin, a cancer-specific anti-apoptotic protein in prostate cancer
progression. The combination of conditional Pten deletion and luciferase-expressing
mouse model allowed us to non-invasively follow the tumor growth through
bioluminescence imaging, permitting the collection of tumors at either the androgen-
dependent (AD) primary growth phase or the recurrent phase when castration-
resistant prostate cancer (CRPC) is formed after initial regression from androgen-
deprivation therapy. Utilizing cell surface markers shown to enrich normal prostate
epithelial stem cells (Lin
-
Sca-1
+
CD49f
+
), we were able to further restrict the parameters
of selection to enrich putative cancer stem cells (CSCs) from both androgen-dependent
and castrate-resistant prostate tumors. These populations of cells exhibited the ability
to self-renew and differentiate to multiple cell types in vitro and in vivo, in concordance
to known characteristics of normal prostate tissue stem cells. Along with the detection of
expression of certain expected stem cell-like markers, evidence was obtained that the
CSCs from the model also retain a high level of survivin expression like the cancer cells.
We demonstrated the contribution of the cancer-associated fibroblasts (CAFs) of the
tumor microenvironment in enhancing the putative cancer stem cells’ self-renewal and

xiii

differentiation potentials. We also found that in vivo growth and differentiation of CSCs
from CRPC cancer were better supported by CAFs derived from CRPC cancer
compared to those from AD cancer. These results suggested that signaling proteins that
are secreted by stage-specific CAFs might support and potentiate the stemness and
tumorigenic properties of the corresponding CSCs. This novel finding deserves to be
further investigated, particularly since stromal fibroblasts remain as an understudied
cellular compartment of the prostate cancer.
Our examination of the role of survivin in prostate cancer was achieved through
the generation of a prostate epithelium-specific double knock-out mouse model lacking
Pten and Survivin. With Survivin deletion alone, normal prostate organogenesis and
growth as well as fertility of mice were not found to be impaired. Survivin homozygous
deletion in conditional Pten knockout mice, however, resulted in the delay of prostate
cancer progression in the sense that even up to 52 weeks of observation, no
adenocarcinoma but only premalignant lesions, namely prostatic intraepithelial
neoplasms (PINs) were detected. This was in contrast to adenocarcinoma formation by
36 weeks in many animals of the control tumor group or the heterozygous Survivin
deletion group. Prostate tumors lacking survivin appeared to display enhanced
apoptosis, decreased proliferation index, increased senescence, and a high degree of
hypertrophic cells, some of these characteristics being atypical of the usual PIN lesions.
Our findings from this direct in vivo genetic study demonstrate the importance of
survivin in prostate cancer progression potentially via its anti-apoptotic and cell division
regulatory roles, as supported by previous studies, as well as open up possibilities of

xiv

survivin’s association with cellular senescence and cancer stem cells that remains to be
explored in the future.

1
CHAPTER ONE: INTRODUCTION
1.1 Prostate cancer
Prostate cancer is the most common lethal malignancy affecting men in the
Western world. It develops as a result of cellular homeostasis disruption in the prostate.
Androgens, the male sex steroids, are important for development and maintenance of
normal prostate, as well as for prostate cancer cell growth and proliferation. Androgens
bind to androgen receptors (AR) located in the cytoplasm of secretory epithelial cells
and stromal cells in the prostate to achieve their effects. AR belong to the steroid
hormone receptor transcription factor family. Upon ligand binding, they become
phosphorylated and dimerized and translocate to the nucleus to bind to androgen
response element within the DNA to activate genes involved in proliferation,
differentiation, apoptosis and secretion (1-2). Prostate cancer development is slow,
taking a course of 20 to 30 years (3), starting with prostatic intraepithelial neoplasia
(PIN) stage, progressing to adenocarcinoma in situ, invasive adenocarcinoma, and
finally, metastatic cancer (4). When detected early, prostate cancer can generally be
cured by surgery and radiation therapy. Advanced stages of this disease are treated
with androgen ablation therapy that initially results in tumor regression. Most common
modes of androgen depletion are surgical (removal of one or both testes) or medical
(administration of antiandrogens and/or leutenizing hormone-releasing hormone
agonists) castration (5). However, in most cases, the tumors become resistant to
androgen deprivation over time and relapse to more aggressive and fatal metastatic
state, known as androgen depletion independent cancer (6-9), or more recently,
castrate-resistant prostate cancer (CRPC). Several proposed mechanisms of prostate

2

cancer progression in low-androgen environment include AR gene amplification, AR
gene mutations, involvement of co-regulators, ligand-independent activation of AR by
growth factors and other kinases, androgen receptor-independent modulation of cell
survival, and cancer stem cell regeneration (5).
1.2 Cancer stem cells
In recent years, there is increasing evidence to suggest the presence of cancer stem
cells (CSCs) and their importance in the initiation and propagation of cancer. Although
making up only a small fraction of the tumor, CSCs may possess the ability to self-
renew and differentiate into cells that compose, perhaps, the majority of the tumor mass
(10). They also have distinct properties such as the extensive proliferation potential,
multidrug resistance, and maintenance of high level of telomerase expression (11).
Normal stem cells live in a niche made up of critical signaling molecules, such as growth
factors and components of the extracellular matrix, that regulate their self-renewal and
differentiation (12). Similarly, CSCs may also depend on this specialized niche for their
function. This niche may potentially be defined by interactions of CSCs with not only
the cancer cells, but also with local stromal cells via cell-cell interactions and by the
molecules secreted by the various cell compartments and the extracellular matrix.
CSCs in brain tumor were found to reside in vascular niches that when disrupted,
inhibits CSC self-renewal and markedly reduces tumor growth (12, 13). This illustrates
the significance of the surrounding microenvironment on the survival and maintenance
of CSCs. The hypothesis of prostate cancer stem cells is partially derived from the stem
cell model of prostate organization. According to this model, pluripotent stem cells give

3

rise to differentiated basal, secretory luminal, and neuroendocrine cells. Androgen
cycling experiments in rodents (14) found that the prostate gland atrophies upon
androgen deprivation but is able to regenerate when androgen supply is restored and
this cycle of gland involution was repeatable many times. This, along with many
subsequent studies, implies the presence of a stem cell population within the prostate.
The finding that prostate stem cells do not need androgen to survive supports how the
existence of CSCs may be the key to the resistance of androgen withdrawal frequently
observed in late stage of prostate cancer (11).
1.3 Pten deletion mouse model of prostate cancer
PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a tumor
suppressor that is often mutated or deleted in human cancers, including prostate
cancer-- 30% of primary (15, 16) and 63% of metastatic prostate cancers (17). PTEN
exerts its tumor suppressive effect through its phosphatase activity to remove
phosphate groups from PIP
3
and in turn serving as a negative regulator of the PI3K/AKT
pathway that is associated with increased proliferation, survival and metabolism of the
cells (18). Homozygous deletion of Pten results in embryonic lethality; thus, studies of
Pten on prostate cancer has been carried out using conditional knock-out mice.
In our studies, we utilized a Pten deletion mouse model that recapitulates the natural
progression of human prostate cancer. This model was established by combining
ARR2Probasin-Cre (PB-Cre4) mice, in which Cre expression is driven by the modified
rat prostate epithelium specific probasin (PB) promoter (19), with Pten
flox/flox
mice (20)
that resulted in a prostate-specific deletion of the phosphatase region of Pten (exon V).

4

Homozygous Pten deletion in these mice led to hyperactivation of the Akt pathway that
contributes to the enlargement of the cells and increased proliferation. Prostates of
these mice display epithelial hyperplasia (increased number of cells without atypia) by 4
weeks of age and murine PIN (mPIN), proliferation of atypical cells, by 6 weeks.
Characteristics of atypia include enlargement of the nuclei, hyperchromatism,
irregularity of nuclear contour, and inversion of the nuclear to cytoplasmic ratio. Similar
to human prostate cancer, these mice also develop invasive adenocarcinoma, which is
epithelial in origin, as determined by the positive expression of androgen receptor (AR),
a marker of the secretory epithelium. The malignant cells were detected by 9 weeks of
age, invading the surrounding stroma and inducing inflammatory and desmoplastic
response (4). Desmoplastic response is described as stromal cell enlargement with
increased extracellular fibers and transformation from a fibroblastic to myofibroblastic
phenotype (21).
This homozygous Pten deletion mouse model is the first animal model in which a
deletion of an endogenous gene results in metastasis, detected in the lymph nodes and
pulmonary alveolar spaces as early as 12 weeks of age. The mice also respond to
androgen ablation by displaying increased apoptosis and decreased tumor volume
following castration, but later developed androgen-independent cell proliferation as
determined by the higher Ki67 staining and quantification compared to age-matched
wild type controls. Additionally, microarray analysis of mice that already developed
invasive adenocarcinoma revealed similar gene expression profile with human prostate
cancer, such as upregulation of cyclin A, clusterin, PSCA, S100P, ERG-1, and
osteopontin, as well as downregulation of Nkx3.1 and myosin heavy chain 11 (4).

5

The conditional Pten deletion mouse model is then further enhanced by
combining incorporating the conditional expression of luciferase reporter gene into the
system (22). Thus, luciferase expression is activated in cells where Pten is deleted by
Cre/loxP recombination, allowing primary tumor growth to be monitored non-invasively
over time by bioluminescence imaging (BLI). Post-surgical castration, tumor regression
and then recurrence were also able to be detected by the initial decrease and then
increase of the bioluminescence signal, permitting the collection of prostate tumor
samples at various stages of prostate cancer development for analysis. This allowed us
to detect a higher degree of hyperplasia of cells with neuroendocrine differentiation in
recurrent cancer compared to primary cancer, consistent with previous findings in
human prostate cancer. This improved model of conditional Pten deletion mice is
significant for it provides us with the ideal tool to better study prostate cancer
progression at both androgen-dependent and castrate-resistant stages. It is key in our
study of the prostate cancer stem cell population and its microenvironment that I will
discuss in the next chapter.
1.4 Survivin
One of the most overexpressed proteins in a wide variety of cancers is survivin, the
smallest member of the mammalian inhibitor of apoptosis protein (IAP) family. It is
comprised of 142 amino acids, a homodimer containing a single Baculovirus IAP repeat
(a 70-amino-acid zinc finger fold) and a long α-helix at its carboxyl-terminus (23). There
are four alternatively spliced survivin transcripts: survivin-2B--165 amino acids, survivin
3B--137 amino acids, survivin Ex3--120 amino acids, and survivin 2α--74 amino acids

6

(24). Survivin level is controlled in part by post-translational modifications. Survivin
expression peaks during mitosis, in part due to the phosphorylation of survivin on Thr 34
by p34cdc2-cyclin B1 that helps increase its stability (25). Polyubiquitination and
proteasomal degradation of survivin results in low levels of survivin at interphase (26).
Survivin’s high expression at mitosis suggests its role in cell division. During
mitosis, survivin associates with multiple components of the mitotic apparatus, such as
centrosomes, microtubules of metaphase and anaphase spindle, and the midbodies
(27). Survivin homozygous deletion resulted in extreme defects in microtubule
assembly by embryonic day (E) 2.5, manifested by lack of mitotic spindles, failure of cell
division and multinucleation. By E 3.5-4.5, 100% lethality of the mice embryo was
observed (28). Additionally, antibody microinjection studies revealed that survivin
inhibition leads to prolonged metaphase arrest, multipolar mitotic spindle formation, and
chromosome attachment defects (27). These data point to survivin’s important roles in
regulating microtubule stability for proper assembly of the mitotic apparatus as well as
cytokinesis.
Survivin has been found to inhibit apoptosis both via the extrinsic or intrinsic
apoptotic pathways, although it differs from other IAP members in its more selective
preference in preventing cell death induced by mitochondria (intrinsic pathway) over the
death receptors (extrinsic pathway) (29). Survivin has been shown to form a complex
with the upstream mitochondrial initiator caspase-9 in vivo (30) and with Smac/DIABLO,
another mitochondrial apoptogenic protein (31). Even though Survivin does not directly
bind caspases, it seems to exert its anti-apoptotic activities by interacting with other

7

partners in vivo (23). For instance, survivin forms an IAP-IAP complex with XIAP,
another IAP member, resulting in stabilization of XIAP against degradation as well as an
increase in its caspase-inhibiting activity (32).
Survivin has also been purported to be involved in cellular stress response.
Studies have presented evidence of physical interactions in vivo between survivin and
at least three molecular chaperones: Heat Shock Protein-90 (Hsp90), HSP60, and the
aryl hydrocarbon receptor-interacting protein (AIP). Their association may be important
to protect survivin against degradation from proteosomes and prevent apoptosis. The
chaperones may also function to escort survivin to its specific subcellular localization
(23).
Survivin is ubiquitously expressed during embryonic development, but not in
most terminally differentiated normal tissues except thymocytes, CD34
+
stem cells, and
basal colonic epithelial cells (27). Survivin’s unique preferential expression in cancer
over normal tissues makes survivin an ideal therapeutic target in cancer. Its
importance in cancer has been established by numerous studies that show cancer cells’
exploitation of its anti-apoptotic and cell cycle regulation functions to facilitate their
expansion. On this basis, various therapeutic strategies against survivin have been
developed over the years, including vaccines, dominant-negative protein, anti-sense
oligonucleotides, and small molecule inhibitors (33). However, survivin’s complex
interactions with multiple signaling pathways such as HIF-1α and HSP90, PI3K/AKT,
mTOR, ERK, p53, PTEN, Ras, and Bcl-2 signaling pathways (34) suggests a need for
more extensive studies on survivin to fully decipher its complete functions in tumors

8

aside from the well-known inhibition of apoptosis and cell cycle regulation, as well
identification of its partners for more effective targeted therapy.
1.5 Hypotheses and experimental strategies
Androgen deprivation therapy through chemical or surgical castration is the
current main mode to treat advanced prostate cancer. Although initially the prostate
tumor regresses in response to androgen withdrawal, eventually the tumor gains
resistance and relapses even with depletion of normal androgen level that is found to be
required for normal prostate cell growth and maintenance. The recurrent cancer is
usually aggressive, metastasizing preferentially to the bone (6-9). What is then
responsible for this recurrence of prostate cancer? Scientists have been struggling with
this question for some time, coming up with some possible explanations that have
arisen through extensive studies in this area over the years, including the presence of a
rare cancer stem cell population. The idea of their existence was based on the
characteristics of normal prostate stem cells that are shown to express low levels of
androgen receptor and do not rely on androgen for their quiescence, proliferation, self-
renewal, and differentiation (7, 9). Can there also be a small population of cancer stem
cells that can survive in the low-androgen environment and possess the ability to self-
renew and differentiate to cells that make up the majority of the tumor? Supported by
our ability to follow progression, regression and relapse of prostate cancer in living mice
in the conditional Pten deletion model of prostate cancer, along with acquired
experience in obtaining cultures of enriched subpopulations of epithelial, fibroblastic and
cancer “stem cell-like” cells (CSCs) at specific stages of growth or re-growth of the

9

tumor, we were set to begin a critical analysis of the important hypothesis that CSCs are
required for growth, progression, metastasis, and recurrence of prostate cancer. This
hypothesis is based on the cues in the field that while the bulk of the tumor cells are
differentiated with only a limited proliferation and tumorigenic potentials, only a minor
subpopulation of cells that resides within the tumors is capable of both self-renew and to
give rise to differentiated tumor cells. Key components and interactions that may
regulate genesis, survival, self-renewal and differentiation are likely to be present in the
tumor microenvironment in the forms of secreted factors, cell-cell contacts and cell-
matrix adhesions. Our focus is placed on studies to better understand the genesis,
biology and behavior of CSCs in response to soluble factors secreted by the fibroblastic
stromal cells present in the tumor microenvironment. Although it is widely recognized
that stroma is important for survival, motility and growth rates of the prostatic epithelium,
no previous reports, other than ours (39) have linked stroma to CSC activity.
Characterization of paracrine factors released by stage-specific stromal fibroblasts that
regulate prostate CSC activity is expected to offer new directions for therapeutic
development against a cellular compartment different from the malignant epithelium.
Cell sorting using cell surface markers that have been shown to isolate normal
prostate stem cells, Sca-1 and CD49f (35, 36), was crucial for our goal to identify a
putative CSC population. In vitro spheroid formation assay using three-dimensional co-
culture system and in vivo renal capsule transplantation assay were key in examining
the candidate cancer stem cells’ self-renewal and differentiation potentials as well as
the influence of various supporting stromal cells on the putative CSCs. Histological,
immunohistochemical, and immunofluoresence analyses were important in determining

10

the morphology and cell composition of the spheroids and glandular structures formed
by the putative CSCs. Real-time quantitative PCR was utilized to elucidate the
expression profile of the CSCs. The progress and results obtained to date for this
project are outlined in Chapter 2.
Survivin is one of the most universally overexpressed proteins in a wide range of
different cancers. Its unique expression in cancers and not in most normal adult
differentiated cells makes it an attractive therapeutic target (27). Our previous studies
on the role of bone morphogenetic protein 7 (BMP 7) in prostate cancer led to the
conclusion that BMP7 exerts its protective effect from apoptotic stimuli in human
prostate cancer cell line C4-2B through upregulation of survivin (37). We also reported,
for the first time, that survivin expression in C4-2B is regulated by the transcription
factor Runx2, which is induced in increasing levels of BMP-7 (38). In the conditional
Pten deletion model of prostate cancer, BMP-7, Runx2, and Survivin protein levels all
increase with tumor growth (37, 38). Thus, we wished to study the biological
consequence of loss of survivin function in the prostate epithelium. It was, however, not
known whether survivin has a role in the development of a normal prostate. Since we
detected practically no expression of survivin in the growth of the normal adult mouse
prostate whose development is primarily post-natal and associated with sexual maturity,
we hypothesized that homozygous inactivation of Survivin alleles in the epithelial cells
of the prostate would lead to generally normal and fertile animals. After genetically
confirming this fact through our experiments, the role of survivin in prostate cancer
genesis and progression was then tested using the conditional biallelic Pten deletion
model by crossing the tumor model with the floxed survivin allelic mice. Our hypothesis

11

was based on the contention that survivin expression in prostate cancer is a mechanism
of selective advantage for the cancer cells to evade apoptosis and that prostate
epithelium-specific survivin nullizygous condition is likely to enhance cellular apoptosis
to counter prostate tumorigenesis and/ or progression to recurrent cancer. In addition,
we projected that this combined model should be valuable to evaluate the self-renewal,
survival, and differentiation potentials of a fraction of CSCs that may potentially be
deficient in survivin levels. It would be important to determine if subpopulations of CSCs
from the prostate tissues of the combinatorial model may harbor both Pten and Survivin
deletions, as footprints of potential origin from differentiated epithelial cells. To date, we
examined the effect of monoallelic and biallelic inactivation of Survivin by histological,
immunohistochemical, and protein expression analyses of prostate samples at various
time points in the progression of the primary prostate tumor. The results reported in
chapter 3 support previous findings on survivin’s cytoprotective and cell cycle regulatory
functions in cancer cells, and also shed light to some new intriguing observations for
future studies.

12

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15

CHAPTER TWO: STUDY OF CANCER STEM CELLS AND CANCER-
ASSOCIATED FIBROBLASTS FROM TUMORS OF A MOUSE MODEL
OF PROSTATE CANCER

2.1 Abstract
There is evidence that the interactions between cancer cells and cancer-associated
fibroblasts (CAFs) may positively regulate the proliferation and tumorigenicity in prostate
cancer. In this study, we investigated whether CAFs may regulate the biology of prostate
cancer stem cells (CSCs), using a conditional Pten deletion mouse model of prostate
adenocarcinoma to isolate both CAF cultures and CSC-enriched cell fractions from the primary
tumors that were formed during the androgen-dependent (AD) phase of growth and castrate-
resistant (CR) tumors that relapsed after androgen ablation. CSCs that were isolated
possessed self-renewal, spheroid-forming, and multipotential differentiation activities in tissue
culture. The biological properties of putative AD-CSCs were determined to segregate with a
cell fraction exhibiting a signature expression phenotype, including Sca-1 (high), CD49f (high),
CK5 (high), p63 (high), survivin (high), Runx2 (high), CD44 (low), CD133 (low), CK18 (low),
and androgen receptor (low). Complete expression profile of CR-CSCs still remains to be
determined. The spheroid-forming efficiency of the CSCs was differentially influenced by the
nature of fibroblasts in a co-culture system: compared to mouse urogenital sinus mesenchyme
or normal prostate fibroblasts, CAFs enhanced spheroid formation with the spheroids
displaying generally larger sizes and more complex histology. Graft experiments showed that
AD-CSCs admixed with AD-CAFs produced prostatic glandular structures with more numerous
lesions, high proliferative index, and tumor-like histopathologies, compared to those formed in
the presence of normal prostate fibroblasts. Interestingly, CSCs obtained from castrate-

16

resistant prostate cancer (CRPC) mice showed enhanced spheroid-forming capacity in vitro
and increased prostatic glandular structures formation in vivo in the presence of CAFs isolated
from androgen-depleted state (CR-CAFs) compared to AD-CAFs. Together, our findings
suggest that signaling proteins secreted by the CAFs that are present within the primary and
castrate-resistant prostate tumors can support and potentiate the stemness and tumorigenic
properties of the CSCs derived from the same tumors. This novel finding deserves to be
further investigated, particularly since stromal fibroblasts remain as an understudied cellular
compartment of the prostate cancer. Although it is widely recognized that stroma is important
for survival, motility and growth rates of the prostatic epithelium, no previous reports have
linked stroma to CSC activity. Characterization of paracrine factors released by stage-specific
stromal fibroblasts that regulate prostate CSC activity is expected to offer new directions for
therapeutic development against a cellular compartment different from the malignant
epithelium.
2.2 Introduction
The prostate carcinoma or carcinoma tissue, in general, consists of malignant
epithelial cells and their progenitors, a variety of stromal cells including fibroblasts (and
myofibroblasts), endothelial cells, pericytes, and inflammatory cells. Extracellular
proteins secreted by these different cell types are present in the tumor
microenvironment. These constitute a complex array of growth factors, cytokines,
chemokines, and adhesive molecules that are likely to alter the balance between
proliferation, survival, differentiation, and quiescence in both cancer cell populations and
their progenitors during tumor progression, metastasis or recurrence (1-4). Considering

17

the practical difficulties in obtaining human tissue materials representing different stages
of the disease progression, the mouse models of prostate adenocarcinoma offer a great
opportunity to examine the roles of the various extracellular proteins.
Stromal cells are known to stimulate epithelial cell growth through their ability to
produce extracellular matrix and by secretion of growth factors and cytokines, and to
support angiogenesis (1, 2, 5). There is evidence that fibroblastic cells from prostate
tumors, termed as “cancer-associated” fibroblasts (CAFs) can enhance the tumorigenic
potential of the epithelial compartment (6, 7). Over the past five years, in the field of
solid tumors, an enormous attention has been placed on the study of cancer stem cells
(CSCs). CSCs are considered to be a minor population of tumor-initiating or tumorigenic
cells within the tumor that can self-renew while simultaneously giving rise to tumor cells.
Their stem cell-like properties may be responsible for solid tumor initiation,
homeostasis, progression, metastasis and recurrence (8-11). Since bulk of the tumor
cells have only limited proliferation ability and are non-tumorigenic, CSCs might be
central to the mechanisms in the cancer. Recent studies in solid tumors have shown the
findings of such cells in brain, breast, colon, lung, liver, pancreas, ovarian, head and
neck, melanoma and prostate cancers (9, 12-19). Considering the known role of
fibroblasts of the tumor microenvironment in cancer, it is now important to ask if these
cells do serve critical functions in the biology of CSCs.
Supported by our ability to follow progression, regression and relapse of prostate
cancer in living mice (20) along with the ability to obtain primary cultures of CAFs at
specific stages of growth of the tumor (21), we are set to begin a critical analysis of the

18

effects of CAFs on CSCs isolated from the same tumors. In this chapter, we first
describe isolation and characterization of a small subpopulation of epithelial cells,
enriched for putative CSCs from prostate adenocarcinomas of the conditional Pten
deletion mouse model (20), and then demonstrate that CAFs isolated from these tumors
can significantly support and potentiate the stemness and growth properties of the
CSCs present in the isolated epithelial subpopulations. The study was further extended
to examine the characteristics of the CSCs derived from the CRPC tumors of the same
model.
2.3 Materials and Methods
Animals. The conditional Pten deletion mouse model with simultaneous activation of
the luciferase reporter (cPten
-/-
L), used in the current work was described previously
(20). For tissue grafting, NOD.SCID mice purchased from NCI-Frederick, were used.
Cell sorting. Single cell suspensions from minced prostate tissues were obtained
following the published protocol (20). For magnetic cell sorting (MACS), the cells were
stained with biotinylated “Lin” antibodies (against CD45, CD31 and Ter119; BD
Bioscience; 0.1 g / 10
6
cells) for 10 minutes on ice. After washing with the cell staining
buffer, Lin
-
cell fraction was separated from Lin
+
cells using the DYNAV CELLection™
Biotin Binder Kit (invitrogen) following the manufacturer’s protocol. Lin
-
Sca-1
+
cells were
separated from other cells in the Lin
-
fraction using the same kit and biotinylated Sca-1
antibody (Biolegend). For fluorescence-activated cell sorting (FACS), cells were
stained with biotinylated Lin antibodies followed by PE/Cy5-conjugated streptavidin
(Biolegend; 0.2 g / 10
6
cells), PE/Cy5-conjugated Sca-1 antibody (Biolegend; 0.1 g /

19

10
6
cells), and PE-conjugated CD49f antibody (Biolegend; 0.25 g / 10
6
cells). Stained
cells were then examined using BD FACSAria™ Cell Sorting System with BD
FACSDiva™ software.
Assays for spheroid formation. Culturing and passaging conditions were adapted
and modified from published protocols (22-24). Briefly, sorted prostate

cells were
counted and suspended in 1:1 Matrigel (BD Bioscience) / PrEGM (Lonza) in a total
volume of 250 µL. The mixture was

placed in a well of a 24-well plate, solidified at 37 ℃,
and then cultured in PrEGM. Stromal cells were seeded inside an insert, pore size: 8.0
m (BD Bioscience) above the matrigel. This mixture was cultured at 37 ℃, and half of
PrEGM was changed every 3 days. Spheroids were counted at 14 days after plating.
For serial passages, spheroids formed in Matrigel were digested in 500 µl of dispase
(BD Bioscience)

at 37 ℃ for 30 mins followed by treatment with DMEM/F12 medium
(Invitrogen) containing 10% fetal bovine serum (FBS), collagenase (Sigma; 1 mg/ml),
hyaluronidase (Sigma; 1 mg/ml) and DNase I (Sigma; 1 g/ml) for 30 mins and then in
0.05% Trypsin/EDTA for 10 min. After passing through a 40-µm

filter, cells were
counted and re-plated.
Stromal cells. UGSM was isolated following published procedures (22, 25) . NPFs and
CAFs were isolated as described before (21). The passage number of stromal cells
used in this work was limited to 3-7 for UGSM and 3-10 for NPFs and CAFs.
Immunostainings. The Matrigel layer in the wells harboring spheroids was covered
with OCT, sectioned (8 µm thickness) at -25 ℃, and stained by either H&E or

20

immunofluorescence (20). Primary antibodies against p63 (1:100; Santa Cruz), CK8
(1:100; TROMA-1 antibody; Developmental Studies Hybridoma Bank, University of
Iowa), CK5 (1:1000; Covance), androgen receptor (AR; 1:100; Santa Cruz), Ki67
(1:400; Vector Laboratories), Nkx3.1 (1:400; Abcam), Cre (1:1000; Covance) or
Vimentin (1:50; Cell Signaling) were used. Immunohistochemical analysis of parallel
paraffin sections of 4% PFA-fixed tissues was done by a modified avidin-biotin complex
(ABC) technique (20).
Renal grafting. Epithelial cells with or without stromal cells were mixed in 50 µL
neutralized rat tail collagen type I (BD Bioscience) and placed in the middle part of a
well in a 12-well plate. The grafts were cultured in Bfs medium (26) overnight at 37 ℃
prior to transplanting under the renal capsules of 8 to 12 week-old NOD.SCID male
mice.
PCR analyses. Total cellular RNA (100 ng), extracted by RNAqueous-Micro Kit
(Ambion) was reverse-transcribed by random hexamers using qScript™ cDNA
Synthesis Kit (Quanta), and the reverse transcription reaction (1µL) was then subjected
to PCR amplification using FastStart Universal SYBR Green Master (Roche). PCR
signals were recorded and analyzed in Stratagene MX3000P qPCR system with MxPro
software (Stratagene; v4.01). For DNA quantitative PCR, genomic DNA was extracted
by Pico Pure™ DNA Extraction Kit (Arcturus). DNA sample (100 ng) was mixed with the
primer set and FastStart Universal SYBR Green Master (Roche). PCR reactions were
performed and analyzed using the same qPCR system and software. Primer sets are
listed in Supplementary Table S1.

21

Statistical analysis. All data were presented as means ± SE. Statistical calculations
were done with Microsoft Excel analysis tools. Differences between individual groups
were analyzed by paired t test or chi-square, as appropriate. P values of <0.05 were
considered statistically significant.
2.4 Results
Stem cell-like properties examined in the in vitro cultures
Subpopulation of cells which are Lin
-
Sca-1
+
(LS) cells, selected from the primary
prostate tumors (T) of the mouse model or the prostatic proximal region of normal (N)
prostates were tested for their growth in a modified Matrigel colony assay system (27,
28). As illustrated in Fig. 2-1A, epithelial cells (10
4
) were mixed with 50% Matrigel in the
well, and primary stromal cells (10
4
), namely, UGSM, NPF or CAF, were seeded into
the inserts placed above the Matrigel layer. Spheroids formed from T-LS

cells, in
general, appeared to be larger than the spheroids from N-LS cells (Fig 2-1B; Fig. 2-2A).
Frequently, T-LS spheroids also presented a dense structure in the intra-lumen region
(Fig. 2-1B). While the influence of UGSM and NPFs on the spheroid morphology was
not found to be significantly different from one another, CAFs seemed to exert a
profound effect on the spheroid morphology as seen by their larger, denser and
complex appearance (Fig. 2-1B, C and D). This effect of CAFs was seen on both N-LS
and T-LS cells. N-LS spheroids grown with UGSM or NPFs had two distinct cell layers
containing either p63
+
cells or CK8
+
cells (Fig. 2-1D, top). The sizes of N-LS spheroids
co-cultured with CAFs were generally larger than those formed in the presence of
UGSM or NPFs. Cells with co-expression of p63 and CK8 were found to be increased in

22

T-LS spheroids compared to N-LS spheroids (Fig. 2-2B). T-LS spheroids co-cultured
with CAFs showed not only enlarged size and p63 and CK8 double positive cells in the
inner cell layer, but also multiple layers of CK8
+
cells in the intra-lumen region (Fig. 2-
1D, bottom). Additionally, it was noted that the proportion of CK8
+
cells was increased in
the spheroids of either N-LS or T-LS origin by CAFs as compared to NPFs, and that in
T-LS spheroids p63
+
cell proportion was decreased in the presence of CAFs as
compared to NPFs while in N-LS spheroids the effect was opposite (Fig. 2-2B).
Together, the results suggested that LS cells in the tumor model, like the normal
prostate-derived LS cells might have the multi-potentiality to differentiate to other cell
types, along with a unique attribute to form tumor-like glandular structures in vitro,
especially when exposed to paracrine signaling molecules from CAFs.

23

Figure 2-1. Analyses of spheroids formed from putative normal and cancer stem

cells. A. Illustration of the placement of an insert (pore size: 8.0 μm) containing stromal
cells on top of the Matrigel for the co-culture experiments (E: epithelial cells; S: stromal
cells). B. Phase contrast images of representative spheroids formed from either N-LS
or T-LS cells after 14 days of co-culture with primary stromal cells from different
sources(N: Normal, T: Tumor, LS: Lin
-
Sca1
+
, UGSM: Urogenital sinus mesenchyme,
NPF: Normal prostate fibroblasts, CAF: Cancer-associated fibroblasts). Bar, 100 μm.
C. Illustration of the different sizes of the spheroids formed from tumor LSC cells after
co-culturing with either NPFs or CAFs (LSC: Lin
-
Sca1
+
CD49f
+
). Bar, 100 μm. D.
Comparative immunohistochemical analysis of spheroids formed from normal and
tumor LS cells co-cultured with UGSM, NPFs or CAFs. Sections of spheroids were
analyzed by co-immunofluorescence using antibodies against the basal cell marker p63
(red) and luminal cell marker CK8-Cytokeratin8 (green). DAPI was used for labeling cell
nuclei. Bar, 25 µm.

24

Fig. 2-2. Further characterization of spheroids formed. A. Determination of spheroid
sizes. The diameter of each spheroid was measured using ImageJ software, and the
average size was computed. The number (n) of spheroids counted in each group is
indicated. B. Percentages of p63
+
, CK8
+
and double positive cells detected in the
spheroids. Sections of 3 spheroids in each group were examined using co-
immunofluorescence staining and analyzed under the fluorescence microscope. The
number of positive cells was divided by the total number of DAPI

stained nuclei to
determine the percentage. C. Confirmation of differential efficiency of CAFs and NPFs
in promoting spheroid formation from T-LSC cells. Similar to the results shown in Fig. 2-
3C, the two repeat experiments (repeat 1 and repeat 2) also indicate formation of a
higher number of spheroids in the presence of CAFs as compared to NPFs. In all
panels, statistical significance of the difference between a marked pair is indicated by *,
for p<0.05, or
●
, for p<0.01.
Along with the selection for Sca-1 marker, single cells from normal proximal
prostatic tissues or tumors were also enriched based on another putative murine stem
cell surface marker, CD49f (27). Cells separated by Lin (Fig. 2-3A, left) showed the
percentage of Lin
-
cells in the whole input cell portion to be 78.8% in normal prostatic

25

tissue that was collected from a mouse of 12 months of age, and 45.4% in the prostate
tumor from an age-matched cPten
-/-
L mouse. The Lin
-
fraction was then sorted by the
expression levels of Sca-1 and CD49f. The Sca-1 and CD49f double positive cells were
named LSC cells and the total remainder as LSC
-
cells (Fig. 2-3A, right). The
percentage of LSC cells in the Lin
-
cell population in this normal prostatic tissue was
8.6% (7.27±4.85%, mean from 6 animals) while that in the tumor counterpart was
45.5% (52.37±7.74%, mean from 7 animals), a seven fold increase (p<0.01) in the
tumor tissue (Fig. 2-3B). When LSC or LSC
-
cells isolated from the same tumor were
assayed, the spheroid-forming ability of T-LSC cells, not T-LSC
-
cells, could be
significantly increased when co-cultured with stromal cells (Fig. 2-3C). The efficiency of
spheroid-formation by T-LSC cells increased by about 3-fold in the presence of normal
primary stromal cells, UGSM (p<0.01) or NPFs (p<0.05), but by 5.4-fold when T-LSC
cells were grown with CAFs (p<0.01). There was no significant difference between
UGSM and NPFs in this stimulatory effect. Similar results were obtained from the
analyses of T-LSCs from two other tumors comparing the effect of NPFs and CAFs
(p<0.05) as shown in Fig. 2-2C. For the analysis of self-renewal ability, spheroids
formed from T-LSC cells were dissociated and reseeded in fresh matrigel keeping the
input epithelial and stromal cell numbers the same. This serial propagation was
repeated another four times. The differences in the influence of UGSM, NPFs or CAFs
on spheroid formation in these subsequent generations (G2 to G5) were variable.
UGSM and NPFs were better than CAFs at G2 and G3, and CAFs were approximately
equal to NPFs but better than UGSM at G4. Compared to UGSM or NPFs, CAFs
appeared to be more efficient at G5 (Fig. 2-3D). At G4 and G5, the number of spheroids

26

formed declined from that in G3 when grown with UGSM or NPFs. However, the T-LSC
cells with CAFs displayed a continued trend in increase with serial passages. While
stromal cells, in general, enhanced the spheroid-forming efficiency of T-LSC in the first
generation, it remains to be determined if T-LSC alone may exhibit a different growth
potential at subsequent generations.
We isolated a subpopulation of LSC that expressed highest levels of CD49f,
which we marked as LSC
hi
and the remainder as LSC
me
denoting a medium level of
CD49f expression (Fig. 2-4A, right). The FACS plot on the left of Fig. 2-4A showed the
gating that was used to demarcate LSC
-
cells. In this tumor collected from a 12-month-
old mouse, T-LSC
hi
cells constituted 2.3% of the T-Lin
-
cell population. Analysis of
results from 6 normal prostate and 11 prostate tumors indicated that the content of
LSC
hi
cells increased from 0.73±0.38% in normal to 3.39±1.68% in the tumor
subpopulation (Fig. 2-4B). T-LSC
hi
, not T-LSC
me
, cells were found capable of forming
spheroids. The spheroid-forming efficiency of T-LSC
hi
that was grown with UGSM cells
significantly increased to 2.4- and 5.0-fold when UGSM was replaced by NPFs and
CAFs, respectively (p<0.01) (Fig. 2-4C).

27

Figure 2-3. Spheroid formation analysis with the LSC cell subpopulations. A.
Illustration of the FACS plots used for the isolation of LSC and LSC
-
cell subpopulations.
Cells isolated from normal (N) and tumor (T) prostates were first segregated by Lin
markers (left), and Lin
-
cells were further separated by the expression levels of Sca-1
and CD49f (right). Cells with the Lin
-
Sca-1
+
CD49f
+
phenotype (Q2) were labeled as
LSC; others (Q1, 3, and 4) as LSC
-
. B. Comparison of the proportion of LSC cell fraction
in Lin
-
cell subpopulation between normal and tumor prostates. C. Analysis of the
spheroid-forming abilities of T-LSC and T-LSC
-
cells in the 3D co-culture system with
stromal cells of different sources. T-LSC (10
4
) or T-LSC
-
(10
4
) cells, isolated from the
same tumor tissue were cultured with the same number of UGSM, NPFs, or CAFs for
14 days. D. Spheroids generated from T-LSC cells were passaged for 5 generations,
each time in the presence of the same type of stromal cells as indicated. In panels B
and C, statistical evaluation of the difference between two marked groups is indicated
by *, for p<0.05,
●
, for p<0.01, or NS, for not significant.

28

Figure 2-4. Analysis of the T-LSC
hi
cell subpopulation. A. FACS plot showing
separation of T-LSC
hi
or T-LSC
me
cells from the T-LSC cell subpopulation. Unstained
cells were used for gating T-LSC
-
cells in the Lin
-
fraction. Labeled cells with the highest
CD49f and Sca-1 levels were labeled as T-LSC
hi
, and those with medium CD49f
expression levels were T-LSC
me
. B. Comparison of the percentage of LSC
hi
cell
subpopulation in the Lin
-
cell subpopulation between normal (N) and tumor (T)
prostates. Accumulated data from 6 normal and 11 tumored mice demonstrated that the
LSC
hi
cell subpopulation in tumors was 4- to 5-fold higher than that in the normal
prostate. C. Analysis of the spheroid-forming ability with T-LSC
hi
and T-LSC
me
cells co-
cultured with different stromal cells. D. Detection of Pten gene deletion in T-LSC
hi
cells.
Genomic DNA extracted from N-LSC
hi
, T-LSC
-
and T-LSC
hi
cells were examined using
primers specific to Pten exon5 DNA sequence by real-time PCR. Intact Pten allele level
detected in N-LSC
hi
cells was set to 100%. The location of primers inside Pten exon 5
is shown above the bar of T-LSC
hi
cells. In panels B-D, statistical significance of the
difference between a marked pair is indicated by *, for p<0.05,
●
, for p<0.01.

Gland-forming potential of the cell subpopulations.
To determine the tissue regeneration characteristics, grafts were produced by
mixing T-LSC
hi
, T-LSC
me
or T-LSC
-
cells with UGSM cells for transplanting under renal
capsules. The H&E staining of the representative tissue sections from grafts collected

29

after ten weeks are shown in Fig. 2-5A. Grafts from T-LSC
hi
cells displayed prostatic
glandular structures containing multiple compact cell layers mimicking a tumor-like
histology. Cells located in the structures showed enlarged nuclei (Fig. 2-5C; H&E). The
incidence of glandular structure formation by T-LSC
hi
cells was 100% (Table 2-1).
However, no such structures were found in grafts formed from T-LSC
me
cells. Two out
of six grafts formed from T-LSC
-
cells contained small glandular structures with multiple
cell layers. IHC staining confirmed that the grafts formed from T-LSC
hi
cells with UGSM
were mostly composed of AR, CK8 and CRE positive cells (Fig. 2-5C; Fig. 2-6C). Since
UGSM cells were prepared from embryos lacking the Cre transgene, any contaminating
epithelial cells therein should also be negative for CRE staining. Of a total of eight
glandular structures from three different grafts examined in details, we detected CRE
staining in all (100%). Practically all cells (98-100%) within the structures stained for
CRE expression (data not shown). Thus, it was considered unlikely that the structures
formed from T-LSC
hi
were derived from contaminating epithelial cells present in the
UGSM preparation. To determine whether T-LSC
hi
cells alone could have tumorigenic
potential, grafts containing only tumor T-LSC
hi
cells (10
4
), without UGSM cells, were
prepared and transplanted into four animals. A single glandular structure was found in
one of these grafts. This lesion in this graft resembled murine prostate intraepithelial
neoplasia (PIN) with multiple layers of cells with enlarged nuclei (Fig. 2-5C; top row).
The majority of the cells inside this glandular structure were AR and CK8 positive with a
few also displaying proliferation (Ki67 positivity).
The in vivo effects of NPFs and CAFs were also examined. Grafts formed from
these cells alone did not contain any detectable glandular structures (Fig. 2-5B). Eight

30

of eleven grafts formed from T-LSC
hi
and NPFs were found to contain glandular
structures (Table 2-1; Fig. 2-5B); their sizes were variable. All grafts (11 of 11)
generated from T-LSC
hi
and CAFs were found to form glandular structures and, like
with NPF, also of varied sizes. However, as illustrated in Fig. 2-6A, the number of these
structures detected within a graft was higher with CAFs (4 to 10) than with NPFs (2 to
4). An estimation of the areas covered by the glandular structures in each graft showed
that the cumulative values were larger in the presence of CAFs than NPFs (p <0.05).
These results are shown in Fig. 2-6B. IHC staining of sections of these grafts for AR,
CK8 and Ki67 are shown in Fig. 2-5C, and for NKX3.1, CK5, Vimentin and CRE in Fig.
2-6D. In general, the expression of AR, CK8, NKX3.1 and CRE was detected in the
majority of the cells within the structures with a small number of cells staining for CK5.
The NKX3.1 staining results implied that the CSC under study could differentiate into
NKX3.1 expressing cells which might be the same as the luminal epithelial cells since
all prostatic luminal cells express NKX3.1. Vimentin-positive fibroblastic cells were
detected mostly outside the structures, although a few could also be detected inside in
the case of T-LSC
hi
+ CAFs structures (Fig. 2-6D). We also detected a large number of
proliferative cells with Ki67 expression in these glandular structures, and the
proliferation index in T-LSC
hi
+ CAFs was found to be approximately 3.5-fold higher
than that in T-LSC
hi
+ NPFs (Fig. 2-6C).

31

Figure 2-5. In vivo tissue regeneration analysis with tumor-derived cell
subpopulations. A. Hematoxylin and eosin (H&E) staining of sections of grafts
generated from different tumor cell fractions with UGSM cells. The dashed line
separates graft from the kidney tissues. Glandular structures are indicated by arrows.
Bar, 100 µm. B. H&E staining of sections of grafts from NPFs alone, CAFs alone, and
T-LSC
hi
cells with either NPFs or CAFs. Arrows mark glandular structures. Bar, 100 µm.
C. Comparison of the glandular structures at higher magnification. Sections were
stained by H&E, and for AR (Androgen receptor), CK8 (Cytokeratin 8), or Ki67, a
profliferation marker. Bar, 25 µm.

32

Table 2-1. Detection of prostatic glandular structures in grafts.
Grafts containing tumor epithelial subpopulation (10
4
) and a type of primary stromal
cells (10
4
) were transplanted under kidney capsules. After 10 weeks, each animal was
sacrificed, the kidney with the graft isolated, fixed, and then thin tissue sections were
stained to determine the presence or absence of microscopically detectable glandular
structures in the grafts. Statistical evaluation of the difference in incidence of detection
of glandular structures between a marked pair of individual groups is indicated by *, for
p<0.05,
●
, for p<0.01, or NS, for not significant.
Epithelial subpopulations
Primary stromal
cells
Incidence (%)
- UGSM 0/3 (0)
- NPF 0/3 (0)
- CAF 0/3 (0)
LSC
-
UGSM 2/6 (33)
LSC
me

UGSM 0/5 (0)
LSC
hi

- 1/4 (25)
LSC
hi
UGSM 6/6 (100)
LSC
hi

NPF 8/11 (72)
LSC
hi
CAF 11/11 (100)
●
*
● NS

33

Fig. 2-6. Further characterization of the glandular structures formed in renal
grafts. A. Comparison of the number of glandular structures formed from T-LSC
hi
cells
with either NPFs or CAFs. Tissue sections were stained by H&E, and the number of
detectable glandular structures in each graft was counted. Slides from 8 grafts from the
T-LSC
hi
+ NPFs, and 11 grafts from T-LSC
hi
+ CAFs, which were determined to harbor
glandular structures, were evaluated for this purpose. B. Comparison of the areas
covered by the glandular structures in these same grafts. The areas were measured by
using ImageJ software. The mean ± S.D. value (um
2
) indicated a broad range. C.
Comparison of the proliferative index in the glandular structures formed in the renal
grafts. Ten glandular structures from each group, T-LSC
hi
+ CAFs and T-LSC
hi
+ NPFs,
were analyzed for Ki67 staining, and the percentage of positive cells calculated to
compare the proliferative indices between NPFs and CAFs. D. Further IHC analyses of
the glandular structures. Tissue sections were stained for NKX3.1, CK5 (Cytokeratin 5),
Vimentin or CRE. Black arrow indicates the positive cells which are shown in a higher

34

magnification in the up-right corner. Black arrow head points to cells lacking detectable
NKX3.1 staining. Bar, 25 µm. In panels A-C, statistical significance of the difference
between a marked pair is indicated by *, for p<0.05, or
●
, for p<0.01.

Analysis of selected gene expression and Pten deletion in tumor LSC
hi
cells.
For further characterization of the tumor T-LSC
hi
cells, RNA expression of
specific candidate genes in T-LSC
hi
, T-LSC
me
and T-LSC
-
cells was examined. We
analyzed the expression of markers for the luminal cell (CK18), basal cell (CK5 and
p63), and androgen receptor (AR) in the three subpopulations of cells. Real-time
quantitative PCR values obtained from each PCR reaction were normalized to that of β-
actin. The mean ratio of a given gene expression relative to β-actin in LSC
-
cells was
set as 1 and the expression levels in LSC
hi
or LSC
me
cells were calculated as fold
changes relative to that in LSC
-
cells. The results showed that T-LSC
hi
cells expressed
higher CK5 and p63 but lower CK18, compared to T-LSC
me
cells, which in turn
appeared to express higher levels of CK5, p63 and CK18 than T-LSC
-
cells. Of the
three cell subpopulations, T-LSC
hi
cells also expressed the lowest levels of AR (Fig. 2-
7A). Similar RNA expression patterns were also observed in N-LSC
hi
, N-LSC
me
and N-
LSC
-
cells, indicating that T-LSC
hi
shared traits with their normal counterparts (Fig. 2-
7B). Recognizing that CD44 and CD133 were described earlier as putative surface
markers for human normal and prostate cancer stem cells (19, 28), and CD133, also as
an epithelial stem cell marker in murine prostate (29), we examined the expression of
these genes in the three subpopulations. Contrary to our expectation, we detected
highest RNA levels of CD44 and CD133 in the T-LSC
me
cells followed by T-LSC
hi
and T-
LSC
-
cells (Fig. 2-7C, left). We also analyzed expression of tumor-related genes

35

Survivin, Runx2 and Grp78. We recently described that Survivin and RUNX2 are highly
expressed in both mouse and human prostate cancers (30, 31), and that loss of GRP78
can inhibit the progression of prostate cancer in the Pten deletion mouse model of
prostate cancer (32). A high level of expression of both Survivin and Runx2 genes was
detected in T-LSC
hi
and T-LSC
me
cells compared to T-LSC
-
cells. In contrast, Grp78
expression in the T-LSC
-
cells was higher than in the other two cell subpopulations (Fig.
2-7D), implying that GRP78 might be more relevant to terminally differentiated cancer
cells in the model.

Figure 2-7. Analyses of gene expression in AD-Ca LSC
hi
cells. The RNA expression
levels of different markers in LSC
hi
(hi), LSC
me
(me) and LSC
-
(none)

cells isolated from
either normal (N) or tumor (T) prostate tissues were examined by real-time quantitative
PCR. A. Levels of CK5, p63, CK18 (Cytokeratin 18), and AR expressed in the tumor
subpopulations. B. Similar analysis with the subpopulations from the normal prostate. C.
Analysis of CD44 and CD133 expression in tumor cell subpopulations. D. Analysis of

36

expression of Survivin, Runx2 and Grp78 in the tumor cell subpopulations. In each
panel, statistical significance of the difference in expression level of a gene between a
marked pair is indicated by *, for p<0.05, or
●
, for p<0.01.
Next we attempted to determine the Pten allelic status in the T-LSC
hi
cells. For
this purpose, genomic DNA extracted from tumor T-LSC
hi
and T-LSC
-
cells, and also
from N-LSC
hi
cells derived from a normal prostate of a littermate control animal (with
floxed Pten alleles, floxed L transgene, but no Cre transgene) was subjected to real-
time quantitative PCR. The pair of primers (PtenEX5-forward and PtenEX5-Reverse)
used is located inside Pten exon 5 that is flanked by two LoxP sites. In the event Cre
recombination occurred in all cells of the T-LSC
hi
subpopulation, no PCR product would
be expected. PCR values obtained from each PCR reaction were normalized to that of
tubulin. The mean ratio of the DNA extracted from N-LSC
hi
relative to tubulin was set to
be 100% because these mice did not have Cre gene to induce Pten exon 5 deletion.
The results showed that compared to N-LSC
hi
cells, 43.9±4.2% Pten alleles in T-LSC
-

cells (p<0.01) contained exon 5 and this number reduced to 33.5±2.5% (p<0.05) in T-
LSC
hi
cells (Fig. 2-4D). Thus, it appeared that approximately two-thirds of Pten alleles
lost their exon 5 in T-LSC
hi
cells. It could not, however, be discerned if the majority of
the cells had both alleles or one allele deleted and what might be the deletion status in
the various cell types segregating as T-LSC
hi
cells.
In vitro and in vivo studies of putative CRPC CSCs
We proceeded to examine the spheroid-forming potential of LSC
hi
cells isolated
from conditional Pten deleted mice that developed recurrent prostate tumor after
androgen deprivation through castration. The castrate-resistant tumors, detected by

37

bioluminescence imaging of the cPten
-/-
L mice, were collected at 4-6 months post-
castration. We found that in absence of supporting stromal cells, only CRPC LSC
hi

cells, and not LSC
med
or LSC
-
, have the capacity to form spheroids in vitro (data not
shown). As observed in primary tumors, CRPC LSC
hi
formed more spheroids in the
presence of AD-CAFs than in medium alone. However, CRPC LSC
hi
displayed a
preferential response to stimulation by CR-CAFs over AD-CAFs in the number of
spheroids formed (Fig. 2-8A). Both AD-CAFs and CR-CAFs significantly increased the
size and density of the spheroids formed compared to medium alone (Fig. 2-8B).
Next, we made a comparison between AD-Ca LSC
hi
and CRPC LSC
hi
in their
ability to form prostatic glandular structure in the presence of CR-CAFs in vivo. We
engrafted the mixture of cells and matrigel under the renal capsule of intact male
NOD.SCID mice and harvested the grafts 10 weeks later. Two out of 6 grafts formed
from AD-Ca LSC
hi
and CR-CAFs displayed the formation of glandular structures (Fig. 2-
9A, Table 2-2), confirmed with the positive expression of CK8 and AR, while all four
grafts of CRPC LSC
hi
in combination with CR-CAFs showed the presence of glandular
structures (Fig. 2-9B, Table 2-2). Intriguingly, one out of the four CRPC LSC
hi
grafts
exhibited an undifferentiated morphology (Fig. 2-9B, right) never seen in any of our
previous in vivo renal capsule graft results, along with the usual differentiated glandular
structures (Fig. 2-9B, left). The morphology of the structures formed by CRPC LSC
hi
cells in combination with CR-CAFs (Fig. 2-9B) appeared to be larger and more complex
than those formed by AD-Ca LSC
hi
cells and CR-CAFs (Fig. 2-9A), with more multiple
layers of cells resembling tumor histology.

38

Figure 2-8. In vitro spheroid forming analysis of CRPC LSC
hi
cells. A, Quantitation
of mean number of spheroids formed by CRPC LSC
hi
cells without co-culturing with
supporting cells (medium only) and with the presence of AD-CAFs and CR-CAFs
(CRPC: Castrate-resistant prostate cancer, AD-CAFs: Androgen-dependent cancer-
associated fibroblasts, CR-CAFs: Castrate-resistant cancer-associated fibroblasts).
The blue bars denote average total number of spheroids while the purple bars represent
mean number of spheroids larger than 100 µm. B, Phase contrast images of the largest
spheroid size found in each combination of culture at 100x magnification, after 19 days.
Size of each spheroid is marked by the bar.

39

Table 2-2: Grafts containing glandular structure(s)

10
4
of LSC
hi
isolated from AD-Ca (Androgen-dependent cancer) and CRPC mouse
tumors were mixed with the indicated supporting stromal cells (UGSM, AD-CAFs, and
CR-CAFs) in matrigel and engrafted under the renal capsules of intact or castrated
NOD.SCID male mice for 10 weeks before harvesting the grafts. The grafts were then
fixed, processed, embedded in paraffin, sectioned, and stained to determine the
presence of glandular structures.

40

2-9. In vivo renal capsule transplantation assay of AD-Ca and CRPC LSC
hi
cells.
A, H&E and immunostaining of various markers on sections of grafts containing AD-Ca
LSC
hi
cells in combination with CR-CAFs seeded in renal capsules of intact male
NOD.SCID mice. B, H&E staining of representative kidney graft sections of CRPC LSC
hi

cells and CR-CAFs grown in intact male NOD.SCID mice. C, Histological analyses of
kidney graft sections composed of CRPC LSC
hi
cells in combination with various
supporting stromal cells, UGSM, AD-CAFs, and CR-CAFs, engrafted in castrated male
NOD.SCID mice. All microscope pictures were taken at 400x magnification as noted.
We then investigated the glandular structure forming potential of CRPC LSC
hi
in
the presence of low androgen. To recapitulate this environment, we performed the in
vivo renal capsule transplantation assay in castrated NOD.SCID male mice. We tested
the effect of UGSM, AD-CAFs and CR-CAFs on CRPC LSC
hi
cells’ glandular structure
forming ability. We observed that none of the three grafts containing CRPC LSC
hi
cells

41

and UGSM displayed any glandular structures (Fig. 2-9C, top panel), while 2 out of 11
grafts of CRPC LSC
hi
cells mixed with AD-CAFs and 6 out of 11 grafts mixed with CR-
CAFs showed presence of prostatic glandular structures to a varying degree, confirmed
by the positive expression of prostate epithelial luminal cellular markers AR and CK8.
Glandular structures formed by the mixture of CRPC LSC
hi
cells and AD-CAFS (Fig. 2-
9C, second panel from the top) were fewer in number and smaller in area and showed
less Ki67 positivity compared to those containing CR-CAFs (Fig. 2-9C, second panel
from the bottom). Similar to results obtained from intact NOD.SCID mice, the grafts
containing CRPC LSC
hi
cells and CR-CAFs exhibited the formation of undifferentiated
tumor-like phenotype (Fig. 2-9C, bottom panel) in one out of 11 grafts, while 6 out of 11
grafts showed differentiated glandular structure morphology (Fig. 2-9C, second panel
from the bottom). Table 2-2 shows a summary of the in vivo renal capsule
transplantation assay results discussed in this section.
2.5 Discussion
It is becoming increasingly clear that normal tissue stem cells are localized in a
defined microenvironment that provides specific factors for the maintenance of the
properties of the stem cells as well as for the regulation of a balance between
proliferation, differentiation and quiescence of these cells (33-36). In prostate cancer,
there is strong evidence that signals originating from the cancer-associated fibroblasts
(CAFs) could significantly enhance the tumorigenicity of cancer cells. As a central role
for CSCs is being ascribed for tumor homeostasis and progression (37-39), we wished
to inquire if CAFs may regulate the biology of prostate CSCs. This is a critical question

42

for the hypothesis that terminally differentiated cancer cells may have only limited
proliferation ability and CSCs, with asymmetric division to both self-renew and
differentiate may indeed be responsible for the growth and progression of the tumor. In
this report, we describe a mouse model of prostate adenocarcinoma from which CSC-
enriched epithelial cells were derived to examine the effects of CAFs that were also
generated from the tumors of the same model.
Interest in this study is four-fold. First, a modified method is described for the
isolation of epithelial cell fractions retaining a small number of cells with properties of
putative CSCs. We started our study with primary prostate tumor. A cell fraction from
this tumor model is shown to possess self-renewal and spheroid-forming abilities along
with multipotentiality for differentiation in vitro, and the ability to form tumor-like
glandular structures in vivo under appropriate conditions. The selection for tumor cells
(T-LSC
hi
) with high levels of expression of both Sca-1 and CD49f surface markers
appears to discriminate between these cells from those with high Sca-1 and medium
CD49f levels (T-LSC
me
). While a cell fraction contained in the T-LSC
hi
subpopulation
displays spheroid-forming ability and the capability to generate prostate glandular
structures, the T-LSC
me
subpopulation is practically devoid of these capabilities. The
CSC-enriched T-LSC
hi
is still mostly composed of non-CSC epithelial cells as evident
from the efficiency of in vitro spheroid-forming ability, although it is likely that viability of
all cells in the subfractions may not withstand the steps used for the isolation. The bulk
of the cells in the T-LSC
hi
compartment may represent transit-amplifying cells and
terminally differentiated cells, and, thus, indicating that the markers used like Sca-1 and
CD49f, are shared with non-CSC cells, but still, use of these markers in a quantitative

43

manner, as shown here, does contribute to enrichment of prostate CSCs from the
tumors of this mouse model.
Second, we have observed a significant difference in the pattern of relative
expression of certain relevant genes in the T-LSC
hi
and T-LSC
me
subpopulations. While
expressions of basal cell markers CK5 and p63 are stronger in T-LSC
hi
relative to T-
LSC
me
, the T-LSC
me
fraction is found to express higher levels of CK18 and AR as
compared to T-LSC
hi
, although the level of AR is significantly reduced in both
subgroups in comparison to the T-LSC
-
cells. The same general pattern is found in the
respective subpopulations from the normal mouse prostate. Thus, it appears that CSC-
enriched fraction from the prostate tumor of the Pten deletion model contain cells with
the characteristics of the similarly enriched fraction from the normal mouse prostate.
Two other putative epithelial stem cell markers were examined. The levels of each of
CD44 and CD133 transcripts appear to be significantly higher in T-LSC
me
subpopulation
of the tumors relative to either T-LSC
hi
or T-LSC
-
groups of cells, implying that these two
markers may not characterize the CSCs of the tumors in the Pten deletion mouse
model. We also examined expression levels of three cancer-related genes: Survivin,
Runx2 and Grp78. Survivin, a member of the inhibitor of apoptosis (IAP) protein family,
is highly expressed in human cancer (40). In the conditional Pten deletion mice, we
demonstrated a strong correlation between increased levels of Runx2 transcription
factor with the growth of the tumor (31), an observation that is very similar to what we
described for Survivin protein levels in the same model (30). Moreover, Runx2 appears
to be a major regulator of Survivin gene transcription in prostate cancer cells (31).
GRP78, a major ER chaperone is reported to be highly induced in a wide range of

44

tumors including prostate cancer (41), and we described that loss of GRP78 in the
prostatic epithelium can prevent prostate tumor formation in the Pten deletion model
(32). Here, we find that while Grp78 expression is higher in the T-LSC
-
population
compared to either T-LSC
hi
or T-LSC
me
subpopulations, the pattern is opposite in the
case of Survivin or Runx2. Survivin and Runx2 are expressed in both T-LSC
hi
and T-
LSC
me
groups of cells at levels even higher than the bulk of the cancer cells
represented in the T-LSC
-
fraction. Based on these results, we project that high levels of
expression of Survivin and Runx2 might be associated with both CSCs and transit-
amplifying cells as it is with many cancer cells. However, this contention remains to be
tested at the level of individual cells, a task that is difficult at this time in the absence of
definitive markers for the cell types under study.
Third, for the first time, we demonstrate that the spheroid-forming efficiency of
the CSC-enriched cells is differentially influenced by the fibroblasts in co-cultures. The
modified spheroid-forming co-culture system we used has the promise to be a powerful
method to facilitate the studies of paracrine signalings in interactions between stromal
fibroblasts and CSCs. Because fibroblasts are located on the insert above the matrigel
layer, there is no direct cell-cell contact between the two cell groups in this system. An
important finding from such analysis is that as compared to UGSM or NPFs, CAFs
enhance spheroid formation in the first generation by approximately two-fold. In vivo,
the grafts grown from the T-LSC
hi
cells are found to contain multiple glandular
structures in each case, although grafts formed with CAFs appear to exhibit higher
proliferative index as compared to those formed with NPFs. The observations with

45

CAFs underscore a role of CAFs in CSC biology, and open up possibilities for better
identifying the responsible molecular interactions.
Fourth, the enrichment of putative CSCs was also able to be applied to castrate-
resistant prostate tumors of our mouse model. Like in primary tumors, this
subpopulation of cells showed the capacity to form spheroids in vitro and form prostatic
glandular structures composed of differentiated luminal cells in vivo. However, our
initial findings also suggest some differences in the characteristics of LSC
hi
cells and
CAFs isolated from primary versus castrate-resistant tumors. CRPC LSC
hi
cells
appeared to consistently respond better to CAFs isolated from similar androgen-
depleted environment than AD-CAFs, in vitro and in vivo. This observation supports the
importance of microenvironment in supporting the self-renewal, proliferation, differential
potential, and quiescence of stem cells, including cancer stem cells. CRPC LSC
hi
cells
in combination with CR-CAFs also exhibited the capacity of forming poorly differentiated
tumor in vivo that was never seen in any of our study on LSC
hi
cells isolated from
primary tumor. Poorly differentiated tumors are associated with rapid progression and
bad prognosis, assigned a high Gleason grade corresponding with severity (42). Given
the degree of malignancy of castrate-resistant prostate tumors compared to primary
tumors, more in-depth studies on the difference between the putative CSC populations
and CAFs from these two different environments may offer an explanation on cancer
recurrence in the future.
In summary, our study describes a process refined to enrich the putative CSC
population using the surface marker phenotype of Lin
-
Sca-1
hi
CD49f
hi
from the prostate

46

adenocarcinomas of the Pten deletion model and demonstrates that such cells have the
capacity to form tumor-like structures in spheroids in vitro and grafts in vivo. The CSCs
from primary and castrate-resistant tumors appear to retain properties of normal tissue
stem cells, such as, the potential to self-renew and to generate differentiated progenies.
Most notably, we present evidence that CAFs could enhance both the stemness and
growth potentials of the CSCs. It is likely that these new clues could be further
developed to better understand the biology of CSCs in prostate cancer and potentially,
in cancers, in general.

47

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27. Stingl J, Eirew P, Ricketson I, et al. Purification and unique properties of
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51

CHAPTER THREE: ROLE OF SURVIVIN IN PROSTATE
TUMORIGENESIS

3.1 Abstract
Survivin, a member of the inhibitor of apoptosis protein (IAP) family, is expressed
in most cancers, including prostate cancer. Using the conditional Pten deletion mouse
model, we described earlier that increase in survivin levels parallels the growth of the
prostate tumor (Cancer Res. 66: 4285-90, 2006). Here we first demonstrated that
homozygous deletion of the Survivin gene in mouse prostate epithelium had little impact
on prostate organogenesis and development, or on the fertility of the mature males.
However, when conditional biallelic Survivin deletion was combined with the Pten
deletion mouse model, prostate tumorigenesis was negatively impacted. Groups of
animals at about 8, 20, 36 and 52 weeks of age were investigated for the histopathology
of the prostate. While by 52 weeks most of the animals with wild-type or monoallelic
Survivin deletion displayed adenocarcinoma-like lesions, the most severe lesions seen
in the biallelic Survivin deleted mice only resembled high-grade prostatic intra-epithelial
neoplasia (PIN). Pathology of PINs in this group also appeared different from those of
the other groups. Many atypical cells contained large hypertrophic cytoplasm.
Desmoplasmic reaction in the PINs of the group with biallelic survivin loss was minimal
until the late ages; extensive cellular exfoliation was also noted during early and middle
stages of these lesions. The lesions with survivin loss appeared to indicate a reduced
proliferation index and increased apoptosis. Senescent cells could be detected in the
lesions throughout the time points examined. These characteristics conform to both the

52

histopathologic observations and the known primary cellular functions of survivin in
cytokinesis and protection from apoptosis. Survivin deletion was also associated with
reduction in the protein levels of another IAP member XIAP, and of the activated form of
BCL-2 in the tumors. Together, the results suggest a strong role of survivin in the
progression of PINs to adenocarcinoma, and that survivin interference at the PIN stages
may be a valuable therapeutic strategy to halt or delay further progression.
3.2 Introduction
Survivin, a 142-amino acid protein belonging to family of inhibitor of apoptosis
family (IAP), is considered as a cancer therapeutic target as aberrant high expression of
survivin was documented in many different types of human cancer (1-5). It is thought
that survivin over-expression might allow accumulation of mutations in transformed cells
and thereby promoting tumor progression. Its expression is associated with increased
resistance to cancer therapy-induced apoptosis and with lower patient survival (6).
Survivin contains a single baculoviral inhibitor of apoptosis repeat (BIR) domain and
carboxyl terminal α-helix and takes form as a homodimer. It blocks apoptosis by
interacting with other partners, such as other IAPs like XIAP, instead binding directly to
the caspases which are the effector apoptotic factors (7,8). Phosphorylation of survivin
at Thr 34 by p34cdc2 is essential for its cytoprotective function (9).
Transcription of the Survivin gene that is prominent in the mitosis phase of the
cell cycle is also regulated by various growth factors and cytokines (10, 11). There is
evidence that survivin also exists in the extracellular pool in the tumor
microenvironment, and could be absorbed by the cancer cells for their malignant

53

progression (12). Survivin’s differential subcellular localization indicates its multiple
functions. Cytoplasmic/mitochondrial survivin is associated with a protective role against
apoptosis, whereas nuclear survivin is proposed to be a regulator of cell division (13).
In normal cells its expression is at its highest in the G2/M phase of the cell cycle, but in
tumors, it is reported to be independent of the cell cycle (14,15). Survivin is part of the
chromosomal passenger complex (CPC), composed of the Aurora B-kinase, Borealin,
and INCENP. The CPC acts to ensure proper attachment between the mitotic spindle
and chromosomes and correct sister chromatid segregation, allowing successful
cytokinesis (16). In addition, survivin has been found to stabilize the mitotic spindle and
mediate spindle assembly checkpoint (6, 17).
Germline knockout of survivin gene is embryonic lethal (18), and its conditional
knockout in thymocytes leads to impaired cell proliferation, cell cycle arrest, mitotic
spindle defects and apoptosis (1, 19), in neuronal precursors to perinatal lethality and
apoptosis (20), in endothelial cells to embryonic lethality (21), and in hematopoietic
progenitors to mortality, bone marrow ablation, and erythropoiesis defects (22). Prostate
gland, which is developed mostly postnatally, was not investigated earlier for targeted
disruption of survivin function.
We have previously reported a strong expression of survivin protein in prostate
cancer (23) of the conditional Pten-deletion mouse model (24-26) and in human
prostate cancer specimens (27). Interestingly, we also documented that certain
extracellular signaling proteins, such as bone morphogenetic proteins BMP 2 and
BMP7, continue increasing with the progression of prostate cancer in this mouse model,

54

and that there is a direct relationship between BMP/Smad signaling and survivin up-
regulation (23, 28). Additionally, we have identified Runx2, the master transcription
factor for osteoblast differentiation as a key regulator of survivin transcription in prostate
cancer cells, and observed that BMP signaling is also involved in upregulation of Runx2
protein expression in these cells (23, 27). In this regard, it was interesting to note that in
the conditional Pten deletion model of prostate cancer, protein levels of BMPs, Runx2,
and Survivin all increase with the tumor growth (23, 27, 28), implicating a potentially
central role of survivin in prostate cancer. To determine the extent of survivin
contribution to prostate tumor progression in this model system, we first document here
that prostatic epithelium-specific deletion of Survivin has no significant effect on prostate
organogenesis and function. Based on this finding we proceeded to delete one or both
alleles of Survivin in the Pten deletion model, and through analyses of these new strains
we provide direct genetic evidence that loss of survivin expression in the prostate
epithelium strongly inhibits the progression of prostatic premalignant lesions to
adenocarcinoma in these animals.
3.3 Materials and Methods
Generation of prostate-specific survivin- or survivin plus Pten- deletion mice.
For prostate epithelium-specific Survivin knockout we used floxed Survivin (S)
allelic (S
f/f
) female mice with 129sv/Swiss background (1) to cross with male PB-Cre4
(32) transgenic mice (C57BL/6/ DBA2), yielding progenies with heterozygous or
homozygous deletion of Survivin (cS
+/-
and cS
-/-
, respectively where c depicts Cre).
Double deletion of Pten and Survivin in the prostate was generated by mating S
f/f

55

female mice with male mice carrying the c transgene and Pten
f/f
alleles on
C57BL/6xDBA2/129 background (24). All animals produced in the process were of
mixed genetic background. More detailed breeding schematics for various mouse
genotypes are outlined in Figure 3-2. Four distinct groups were generated: 1) Normal
control group: contained the floxed alleles without c, abbreviated as Pten
f/f
S
f/f
); 2)
Control tumor group: c; Pten
f/f
; S
wild-type/wild-type
or cPten
-/-
S
+/+
; 3) Experimental group
with monoallelic deletion of Survivin: c; Pten
f/f
; S
wild-type/f
or cPten
-/-S+/-
; and 4)
Experimental group with biallelic deletion of Survivin: c;Pten
f/f
; S
f/f
or cPten
-/-
S
-/-
.
Mouse genotyping
DNA was extracted from the mouse tails and/ or prostate tissues and subjected
to PCR to determine the genotype. Cre was detected as a 500 bp fragment, wild type
and floxed Pten generated amplicons in the size of 1 kb and 800 bp respectively, while
wild type and floxed Survivin was differentiated by the PCR products, 386 bp and 577
bp, in that order, detected using primers Adv 25 and Adv 28 (1). Deletion of Survivin in
the prostate was confirmed by the presence of 420-bp fragment generated with primers
Adv17 and Adv28 as described in (1). Cre forward primer:
5′-CTTCTGTGGTGTGACATAATTGG-3′, Cre reverse primer:
5′-GATGAGTTTGGACAAACCACAAC-3′, Pten forward primer:
5’-AAGCAAGCACTCTGCGAAACTGA-3’, Pten reverse primer:
5’-GATTGTCATCTTCACTTAGCCATTGGT-3’.

56

Histopathology
Prostate tissues were collected at four different age categories (about 8, 20, 36,
and 52 weeks) and incubated in 4% paraformaldehyde overnight at 4° C and then
washed twice in PBS for 30 minutes each time the next day before storing them in 70%
ethanol. The fixed tissues were then processed, embedded in paraffin, cut to 5 µm
sections onto microscope glass slides, and subsequently stained with hematoxylin and
eosin after deparaffinization and rehydration (29, 30).
Immunohistochemical analysis
The paraffinized prostate tissue sections were deparaffinized and rehydrated
before subjecting them to antigen retrieval in 10 mM sodium citrate buffer at 95° C for
15 minutes and cooling them for 1 hour at room temperature afterwards. The slides
were then incubated in 3% H
2
O
2
in methanol for 20 minutes to block the endogenous
peroxidase activity and then rinsed three times in TBS, 4 minutes each time. The
sections were blocked with normal goat serum (Vector Laboratories, cat. # S-1000) and
Triton X-100 in TBS for 1 hour at room temperature and incubated in primary antibody
solutions overnight at 4° C. Negative controls were incubated in TBS, without any
primary antibody. Antibodies used: Androgen receptor (Santa Cruz Biotechnology cat.
# sc-816), PTEN (Cell Signaling Technology cat. # 9559, Phospho-Akt (Cell Signaling
Technology cat. # 3787), Ki67 (Vector Laboratories cat. #VP-RM04), p63 (Abcam),
Cleaved Caspase-3 (Cell Signaling Technology cat. # 9661), Survivin (Cell Signaling
Technology cat. # 2808) and Cytokeratin 8 (Development Studies Hybridoma Bank, IA,
U.S.A.). The next day, the slides were washed three times in TBS, 4 minutes each, and

57

then incubated in biotinylated secondary antibody (affinity purified biotinylated goat anti
rat IgG (H+L): KPL cat. # 16-16-12, affinity purified biotinylated goat anti-rabbit IgG
(H+L): Vector Laboratories cat. # BA 1000) for 30 minutes at room temperature. After
another round of washing in TBS, the sections were developed using the Vectastain
Elite ABC kit (Vector Laboratories cat. # PK-6100) according to the product manual.
DAB (DAKO cat. # K3466) was used as a chromogen to visualize the expression of
each protein on the tissue sections. Finally, the slides were rinsed in deionized water,
counterstained with hematoxylin and dehydrated before glass cover slips were placed
over the stained tissue sections (29,30).
Proliferation index
Tissue sections from four samples in each age group were stained with Ki67.
Three random areas of each section were photographed at 400x magnification. Ki67
positive cells and total number of cells in each picture were counted using Image J
software. Proliferation index was calculated as the number of Ki67 positive cells divided
by the total number of cells.
Senescence-associated β-Galactosidase staining
Prostate tissue samples were collected, embedded in OCT, frozen on dry ice,
and sectioned to 8 µm and set on microscope glass slides and air-dried. The sections
were then fixed in 4% PFA for 15 minutes at room temperature and then washed twice
in PBS for 10 minutes each time at room temperature. The slides were stained in β-
Galactosidase staining solution (X-Gal, NaCl, MgCl
2
, Fe II, and Fe III in phosphate
citrate buffer pH 6.0) overnight a 37°C incubator (31). The samples were then washed

58

three times in PBS until no longer yellow, post-fixed in 4% PFA at room temperature for
2 hours, then washed with PBS, 20 minutes per wash. The tissues were counter-
stained with Nuclear Fast Red (Sigma #) for 2 minutes, rinsed under running tap water
for 3 minutes, dehydrated using 70% ethanol (1 minute), 95% ethanol (1 minute), and
100% ethanol twice (1 minute each time), and finally incubated in xylene 3 times (2
minutes each) before mounting medium was added to the slides to hold the cover slips.
Western blot analysis
Prostate tissues were pulverized and then lysed using RIPA buffer (Sigma).
Protein concentration was determined by BCA protein assay method (Pierce). 15 µg of
protein was loaded in each lane and subjected to western blot analysis. Antibodies
used: Survivin (Cell Signaling Technology cat. # 2808), XIAP (Cell Signaling
Technology), Livin (Cell Signaling Technology cat. # 5471), Bcl-2 (Cell Signaling
Technology cat # 2876), cIAP (Santa Cruz Biotechnology cat. # sc-12410), cleaved
Caspase-3 (Cell Signaling Technology, cat. # 9661), and cleaved Caspase-7 (Cell
Signaling Technology cat. #9491). β-Actin (Santa Cruz Biotechnology, Inc.) was used
as loading control.
Statistical Analysis
Statistical comparisons were established using an unpaired, two-tailed t test. A
minimum of three independent analyses were carried out for each experiment.

59

3.4 Results
Murine prostate organogenesis and growth are not impaired by the loss of
survivin in the prostatic epithelium
Survivin gene deletion in the prostate of male mice was established utilizing the
Cre-Lox P recombination technology. Male mice expressing Cre driven by the prostate-
epithelium-specific rat ARR
2
PB promoter were crossed with female survivin floxed (S
f/f
)
mice to yield progenies with S
f/f
, cS
+/-
, and cS
-/-
genotypes (Fig. 3-1A, Fig. 3-2A). The
genotype of each mouse was determined by PCR analysis, showing a 500 base pair
band for Cre (Fig. 3-1B), a 386 bp product for wild type Survivin, and a 577 bp fragment
representing floxed Survivin (1). Deletion of survivin was confirmed by the presence of
a 420 bp band on the gel (Fig. 3-1B). Murine prostates were collected at various time
points of about 8, 20, 36, and 52 weeks, and there was no significant difference
observed in the gross morphology of the prostates between S
f/f
, cS
+/-
, and cS
-/-
mice in
all age groups. All prostates appeared normal and were similar in size (Fig. 3-1C).
Furthermore, histopathological and immunohistochemical analyses did not detect any
abnormalities in the morphology and protein expression pattern of prostatic glandular
structures of mice with single and double Survivin allelic deletion (Fig. 3-1D). The
proliferation marker Ki67 expression level also did not deviate from normal upon
Survivin deletion (Fig. 3-1D). Thus, Survivin deletion appeared not to affect normal
prostate organogenesis and growth. A number of mature male cS
-/-
were used for
breeding and were determined to fertile, with the size of litters produced falling within
the normal range.

60

S
f/f
cS
+/-
cS
-/-
AP AP
DLP DLP
VP
AP AP AP AP
DLP DLP
VP
DLP DLP
VP
C

61

Figure 3-1. Survivin deletion has no effect on normal prostate development. A,
Prostate epithelium-specific Survivin deletion was established by homologous
recombination of Cre driven by the probasin promoter and Lox P sites flanking all four
exons of Survivin. B, PCR analysis of tissue samples obtained from 20-week old mice to
determine their specific genotypes. AP-anterior prostate, VP-ventral prostate, DLP-
dorsolateral prostate. C, Ventral view of prostate lobes shows normal gross morphology
of tissue with single and biallelic inactivation of Survivin. D, Hematoxylin and eosin
(H&E) and immunostaining of androgen receptor (AR), luminal epithelial cell marker
cytokeratin 8 (CK8), and proliferation marker Ki67 on ventral prostate lobes collected
from 20-week old mice show normal tissue morphology, protein expression pattern, and
proliferation rate of prostates devoid of survivin.

62

Figure 3-2. Breeding scheme for prostate-specific Survivin deleted mice. A,
Conditional deletion of Survivin in murine prostate. B, Double conditional knock-out of
Pten and Survivin in murine prostate. The genotypes of the mice outlined with the red
boxes were used for the study.

63

Loss of survivin in prostate of the conditional Pten deletion mouse model inhibits
tumor progression
For this study we established a prostate-specific Pten and Survivin double knock-
out mouse strain as illustrated in Fig. 3-3A. The complete breeding schematic is
outlined in Fig. 3-2B. Mice with the following genotypes, confirmed with PCR analysis,
were included in our study: Pten
f/f
S
f/f
, cPten
-/-
S
+/+
, cPten
-/-
S
+/-
, and cPten
-/-
S
-/-
. The
presence of Cre was ascertained by a 500 bp fragment (Fig. 3-3B), floxed Pten by a
1000 bp band, wild type Pten by a 800 bp fragment (24), and by a 386 bp product for
wild type Survivin and a 577 bp fragment representing floxed Survivin (1). Deletion of
Survivin alleles was assessed by the presence of a 420 bp band on the gel (Fig. 3-3B)
as well as the lack of the corresponding protein on the western blots (Fig. 3-3C).
The gross morphology of the prostate glands of the Pten
f/f
S
f/f
, cPten
-/-
S
+/+
,
cPten
-/-
S
+/-
, and cPten
-/-
S
-/-
mice was found to be markedly different. Starting at 8
weeks, prostates collected from cPten
-/-
S
+/+
mice already exhibited abnormality by their
whitish, denser appearance compared to the translucent, smaller normal prostate.
While cPten
-/-
S
+/-
seemed to exhibit a gross morphology similar to the
cPten
-/-
S
+/+
animals, the gland of the cPten
-/-
S
-/-
mice displayed a normal morphology.
Although by 20 weeks of age, cPten
-/-
S
-/-
prostates no longer looked normal, the
morphology was significantly smaller and less denser than those of the cPten
-/-
S
+/+
and
cPten
-/-
S
+/-
animals at the corresponding age. This pattern continued until the last time
point of observation (52 weeks of age), with cPten
-/-
S
+/+
and cPten
-/-
S
+/-
displaying a
similar extent of enlargement of the prostate, much more so than that of cPten
-/-
S
-/-
(Fig.
3-3D).

64

65

66

Figure 3-3. Loss of Survivin in conditional Pten deletion mouse model delays
prostate tumor progression. A, Double conditional knock-out mice lacking Pten and
Survivin were generated by crossing mice carrying Cre and floxed phosphatase region
of Pten (exon V) with floxed Survivin mice. B,Genotypes of each mice (shown: 8 weeks
old) were specified by PCR analysis of the tissue samples. C, Status of Survivin
deletion was confirmed at the protein level with this representative western blot analysis
of the ventral prostate of mice at 8, 20, 36, and 52 weeks, with β-actin serving as
loading control. D, Ventral view of prostate lobes of various genotypes isolated from 52-
week old male mice displayed the significantly smaller size of prostate tissue of the
conditional Pten knock-out mouse with deletion of both Survivin alleles. E, Histological
analysis of H&E staining of paraffin-embedded prostate tissue sections of mice ages 8,
20, 36, and 52 weeks confirmed that lack of survivin in the prostate of conditional Pten
deletion mice impedes tumor progression.
Histological and immunohistochemical analyses indicated the nature of the
lesions formed in the presence or absence of survivin expression in the conditional Pten
deletion model. By 8 weeks, 3 of 4 of the cPten
-/-
S
+/+
mice displayed high grade

67

prostatic intraepithelial neoplasia (PIN), which could be identified as PIN3 or 4, while
only one out of 4 of the cPten
-/-
S
+/-
and none of cPten
-/-
S
-/-
mice exhibited this phenotype
at this time point (Fig. 3-3E, Table 3-1). At 20 weeks, however, high grade PIN lesions,
PIN 3 and PIN 4, were observed in all prostate glands of cPten
-/-
S
+/-
(Fig. 3-3E, Table 3-
2). Single atypical cells with large heterochromatic nuclei and large cytoplasm were
found in prostate specimens of cPten
-/-
S
-/-
mice between 8 and 20 weeks of age, and
PIN 2 and PIN 3 were detected on some glands after further aging (Fig. 3-3E, Table 3-
3). As early as 8 weeks, one out of 4 cPten
-/-
S
+/+
mice developed early carcinoma
associated with microinvasion, while none such lesions was observed in the cPten
-/-
S
+/-

or cPten
-/-
S
-/-
group at this age. Appearance of early carcinoma lesions was detected in
the single Survivin allelic deletion in the Pten null prostate at 20-week time point. The
incidence and severity of carcinoma (early to adenocarcinoma) progressively increased
up to 52 weeks in both the cPten
-/-
S
+/+
and cPten
-/-
S
+/-
mice (Tables 3-1, 3-2, and 3-4).
In contrast, no indications of early carcinoma or adenocarcinoma were found in the
prostates of the cPten
-/-
S
-/-
mice at any of the time points analyzed. This analysis was
as thorough as possible as multiple sections of each prostatic lobe of these animals
were examined microscopically. The most severe diagnosis that could be assigned to
these mice at 52 weeks was high grade PINs (Tables 3-1, 3-3). Desmoplastic reaction,
characterized by the presence of larger stromal cells with increased extracellular fibers,
was detectable in prostate lesions of the cPten
-/-
S
+/+
and cPten
-/-
S
+/-
mice by 8 weeks
and on, while it was practically absent in cPten
-/-
S
-/-
group until the 52-week time point.
Analysis of normal Pten floxed/ Survivin floxed (Pten
f/f
S
f/f
) control tissue sections at

68

various time points consistently showed the presence of regular prostatic glandular
structures (Table 3-5).

69

70

71

72

Characteristics of expression of cellular markers in the prostate tumors formed in
the conditional Pten deletion mice with heterozygous or homozygous deletion of
Survivin gene
Immunohistochemistry results showed that the prostate cells making up
glandular structures in all four groups of mice stained positive for androgen receptor
(AR) and the luminal epithelial marker cytokeratin 8 (CK8) was detected in all of the
prostate luminal epithelia. Knock-down of Pten specifically in the prostate epithelium
was confirmed by the distinctive lack of PTEN protein staining in that area compared to
the abundance of PTEN expression in the surrounding stroma. Correspondingly, the
level of detection of phosphorylated AKT was elevated in all mouse prostate tissue
sections with conditional Pten deletion, regardless of the status of survivin expression.

73

A significant downregulation in the expression of Ki67, a proliferation marker, was
observed in prostate tissues of cPten
-/-
S
-/-
compared to cPten
-/-
S
+/+
or cPten
-/-
S
+/-
.
Representative results illustrating these observations are shown in Figs. 3-4A, B. The
results of Ki67 staining suggested a role of survivin in cell proliferation. This observation
was also consistent with the finding that the tumor size in cPten
-/-
S
-/-
being strikingly
smaller in comparison to those of either cPten
-/-
S
+/+
and cPten
-/-
S
+/-
(Fig. 3-3D). Staining
of p63, a marker of basal epithelial cells, showed an interesting pattern. The highest
number of p63 expressing cells was found in the prostates of the cPten
-/-
S
+/+
, while the
lowest was in the cPten
-/-
S
-/-
group (Figs. 3-4A). The difference of p63 expression level
appeared to be correlated with the extent of Survivin deletion as well as the severity of
tumors; in cPten
-/-
S
+/+
and cPten
-/-
S
+/-
prostate samples, proportion of p63-positive cells
increased generally in parallel with the severity of the lesions formed , whereas p63
expression in cPten
-/-
S
+/-
tissues appeared higher than that in the cPten
-/-
S
-/-
, although
both cases could be diagnosed as lesions of high grade PIN (PIN4).
Considering that an increased occurrence of apoptotic cells was noted in the
histological analyses of samples from cPten
-/-
S
-/-
group (Table 3-3), we undertook a
further assessment by immunohistochemistry using an antibody against cleaved
caspase-3. We found that the activated caspase-3 expression level was indeed the
highest in cPten
-/-
S
-/-
compared to the other groups (Fig. 3-5B) at ages 36 and 52
weeks. Another noteworthy histologic observation was that the prostatic lesions in the
cPten
-/-
S
-/-
mice frequently contained, in addition of apoptotic cells, other enlarged,
atypical cells indicative of increased incidence of cellular senescence (Table 3-3, as
compared to Tables 3-2, 3-4, and 3-5). This assumption was then confirmed by an in

74

situ assay of senescence-associated β-galactosidase enzyme activity on the frozen
tissue sections, as described (31). It appeared that a higher proportion of cells that
stained positive for β-galactosidase was present in the cPten
-/-
S
-/-
tissues relative to
either cPten
+/-
or cPten
-/-
S
+/+
tissues, especially at 36 weeks (Fig.3-5D).

While areas of
cell senescence, marked by variable expression of β-galactosidase, could be detected
in the lesions of all

groups at 8 to 20 weeks of age, this reactivity was either absent or
rarely encountered in tissues of cPten
-/-
S
+/+
or cPten
-/-
S
+/-
at 52 weeks of age. In
contrast, the reactivity in the tissues from the cPten
-/-
S
-/-
mice could still be readily
detected in samples at 52 weeks.

75

76

Figure 3-4. Expression pattern of cellular markers in prostates of conditional Pten
knock-out mice with heterozygous and homozygous deletion of Survivin. A,
Immunostaining of dorsolateral prostate lobes of 52-week old mice of various genotypes
using antibodies against androgen receptor (AR), cytokeratin 8 (CK8), basal epithelial
marker p63, PTEN, phosphorylated Akt (P-Akt), and Ki67. B, Comparison of
proliferation index assessed by Ki67 ratio of dorsolateral and ventral prostate lobes of
52-week old mice indicates that the degree of Survivin deletion in the prostates
conditional Pten knock-out mice resulted in a corresponding significant downregulation
of proliferation, especially in the dorsolateral lobe. Blue and purple bars denote
dorsolateral and ventral prostates, respectively. P value of < 0.05 is indicated with *.

77

78

Figure 3-5. Effects of Survivin deletion on other IAP family members, apoptosis,
and senescence. A, Representative western blot analysis of dorsolateral prostates of
36-week old mice showed that XIAP level was down-regulated in prostate tissues
lacking both alleles of Survivin, while livin was relatively unaffected. This pattern of
XIAP expression was consistently observed in 8, 20, and 36 weeks old mice, but not at
52 weeks, probably due to presence of more survivin contributed by the increased
levels of the stroma and immune cells at that age. B, Activated caspase-3 staining of
prostate sections isolated from 36 week-old mice displayed enhanced apoptosis
manifested by the higher cleaved caspase 3 expression detected in conditional Pten
deleted prostate tumors lacking single or both alleles of Survivin compared to normal
prostates and tumors with intact Survivin. C, Phosphorylation level of pro-survival factor
Bcl-2 represented by western blot analysis of ventral and dorsolateral prostate lobes of
36-week old mice was upregulated in cPten
-/-
S
+/+
and cPten
-/-
S
+/-
mice, but when both
alleles of Survivin were deleted, phosphorylation of Bcl-2 decreased dramatically. This
was observed at 8, 20, and 36 weeks, but not at 52 weeks. D, Senescence-activated β-
galactosidase staining results showed the most striking increase in senescence in
conditional Pten deleted prostate sample with complete inactivation of Survivin at 36
weeks compared to samples with intact and monoallelic deletion of Survivin.

Effects of survivin knock-out on the expression levels of other IAP member and
Bcl-2 in the tumors formed
Western blot analysis revealed that XIAP, a member of the inhibitor of a (IAP)
family, displays a similar expression level as survivin. It is upregulated upon Pten
deletion and decreases significantly upon complete deletion of Survivin. This
expression pattern, however, was not observed in Livin, another IAP family member.
Although like survivin, livin protein level increases in the absence of Pten, its expression
was not affected when Survivin is knocked down (Figure 3-5A). Phosphorylation of the
pro-survival factor Bcl-2 was found to be upregulated in conditional Pten mouse
prostate with intact Survivin and single Survivin allele deletion, but is downregulated
when both alleles of Survivin were knocked out, indicating that survivin may be required
for phosphorylation of Bcl-2.

79

3.5 Discussion
In the cancer field, survivin stands as a unique member of the IAP family with
essential roles in mitosis, cellular stress response and inhibition of cell death. It was,
however, not known whether survivin plays a role in the development of the normal
prostate and which mechanisms of this multi-functional protein might be relevant to its
role in prostate carcinogenesis. For this purpose, we first determined if the
organogenesis and growth of the prostate gland might be influenced by survivin. Mice
with conditional inactivation of Survivin in prostate epithelium were generated by
crossing mice of our PB-Cre4 line (35) with floxed Survivin mice (1). Through breeding
and analyses of these mice at various ages up to one year, we demonstrate that
homozygous inactivation of Survivin alleles in the epithelial cells of the prostate leads to
fertile males harboring the prostate gland with generally normal gross and microscopic
anatomy. The role of survivin in prostate cancer genesis and progression was then
investigated using the conditional biallelic Pten deletion model (24-26) by crossing the
tumor model with the floxed Survivin allelic mice. Our contention was that prostate
epithelium-specific Survivin nullizygous condition would likely enhance cellular
apoptosis to counter prostate tumorigenesis. Here, using this combined model we
provide evidence that loss of survivin inhibits progression of premalignant lesions to
adenocarcinoma, and that the premalignant lesions exhibiting decreased proliferation
index are composed of atypical cells, many of which exhibit increased hypertrophy,
apoptosis and senescence.

80

Survivin expression was reported to be up-regulated in the early prostate tumor
growth in both the conditional Pten knockout (23) and the TRAMP (36) models. This
implication of survivin in early pathogenesis is corroborated by our observation that
deletion of either single or both alleles of Survivin can influence the phenotype of the
PIN formed at as early as 8 weeks of age. While all groups displayed low grade PIN
formation, none of the four cPten
-/-
S
-/-
and only 1 of 4 cPten
-/-
S
+/-
mice was found to
develop high grade PIN lesions at this age, compared to 3 out of 4 cPten
-/-
S
+/+
mice.

By
52 weeks, 80-100% of the mice with intact or singly deleted Survivin alleles harbored
adenocarcinoma , while none (0/5) of the mice with the homozygous Survivin deletion
developed adenocarcinoma, although high grade PINs were detected. It is unlikely that
the suppression of the progression to adenocarcinoma is due to variation in the mixed
genetic background of the animals, because some of the cPten
-/-
S
+/-
mice developed
adenocarcinoma at age as early as 20 weeks, whereas cPten
-/-
S
-/-
littermates with
similar mixed background were free of cancer even at about one year age. The question
of whether the cPten
-/-
S
-/-
mice might manifest cancer on further aging was examined
very recently. Of the three cPten
-/-
S
-/-
72 week-old males examined, only one was found
to display development of adenocarcinoma in the prostate (data not shown).
With respect to other histopathology parameters, exfoliation and hyperplasia
were abundantly detected in the cPten
-/-
S
-/-
mice, but only rarely in cPten
-/-
S
+/-
or the
control cPten
-/-
S
+/+
group. Furthermore, hypertrophy or enlargement of cells, and
polyploidy or hyperploidy were relatively more frequent in the prostate samples from
cPten
-/-
S
-/-
compared to the other groups

. Survivin deficient cells have been reported to
exhibit multiple nuclei in vitro and in vivo, consistent with the known role of survivin in

81

the regulation of cytokinesis and cell division (1, 18, 37, 38). Another striking difference
between single and biallelic deletion of Survivin was the lack of desmoplasia observed
in the prostate tissue of cPten
-/-
S
-/-
mice younger than the 52 weeks age group that was
prominently present in cPten
-/-
S
+/+
and cPten
-/-
S
+/-
samples from all four age groups
tested. A reduction in the rate of cell proliferation was evident from the significant
downregulation of Ki67 expression in the specimens from the cPten
-/-
S
-/-
animals. This
effect was the most striking at 8 weeks, whether the samples were from the monoallelic
or biallelic Survivin knockout animals. However, with time, the degree of proliferation
became somewhat parallel with the extent of Survivin insufficiency. Another interesting
observation was that Survivin deletion led to a significant decrease in the number of
p63-expressing cells in the lesions. The nuclear protein p63 belongs to the p53 family of
transcription factor and it is a marker of both basal epithelial cells and a subpopulation
of progenitor/stem cells that holds the potential to differentiate to basal, luminal and
neuroendocrine cells of the prostate (39). In a previous study, we described a
subpopulation of putative cancer stem cells isolated from the prostate of cPten
-/-
mice
that displayed an elevated expression of p63 (40). This subpopulation also displayed a
strong expression of survivin. These observations may serve as clues for a potential
role of survivin in proliferation/ differentiation of cancer stem cells as a mode of
promoting cancer progression, a concept that remains to be explored further in the
future.
Another noteworthy point is that we found that the degree of Survivin deletion in
the tumors differentially affected the detectable levels of expression of some other IAP
family members. For example, the expression of XIAP, but not Livin was reduced. This

82

phenomenon could probably be attributed to the function of the complex known to form
between survivin and XIAP that stabilizes XIAP and protects it from ubiquitin-dependent
degradation (7). The survivin-XIAP complex was also described to enhance XIAP’s
capacity to inhibit caspases and to facilitate tumor growth in vivo (7, 41). It was
interesting to note that BCL-2 activation was suppressed in the prostates of the cPten
-/-
S
-/-
, while there was no significant changes in the phosphorylated BCL-2 level between
the prostate tissues of the cPten
-/-
S
+/-
and the

cPten
-/-
S
++
groups. This observation may
imply a possible link between survivin and activation or stability of activated BCL-2 in
prostate tumors, a point that would be of much interest for a mechanistic investigation.
Consistent with previous findings in various other study systems (42-44), we
observed an increase in apoptosis as assessed by the expression level of activated
caspase-3 in the prostatic lesions of the conditional Pten knockout mice lacking both
alleles of Survivin. Interestingly, we also detected a correlation between survivin
expression and senescence in Pten-deleted prostate tumor tissues. By using the
simplified method of staining for senescence-associated β-galactosidase in the tissue
sections (45), we attempted to measure the extent of senescence induction in the
prostate of the various groups of mice. Presence of senescent cells was readily
detected in conditional Pten deleted mice with intact Survivin at 8- and 20-week groups
but only sparsely at later stages, when the tumor had advanced to early cancer or
adenocarcinoma. This finding is consistent with previous reports that senescence may
be an initial barrier in cancer development (46-48), and that senescent cells exist in
premalignant tumors but not in malignant ones (48). The pattern of senescence
observed in the tumor group with monoallelic Survivin loss was not remarkably different

83

from the control tumor group. However, in the case of biallelic Survivin loss, the lesions
from all the early to the late time periods exhibited staining. Since no adenocarcinoma
developed in this group, the presence of stains is consistent with the persistence of
premalignant lesions at time points when the other groups had progressed to cancer.
Although, an association of survivin loss with senescence in the lesions is indicated by
these results, it is, however, unclear whether this relationship is direct or secondary to
the loss of the multi-functionality of the survivin protein that may be critical for the
progression of the preneoplastic lesions to cancer. It is also possible that the defects in
microtubule assembly, loss of mitotic spindles, and formation of multinucleated cells, the
abnormalities that are triggered by the loss of survivin (6, 14, 18) could make the cells
prone for senescence. Further studies into the effect of survivin on cellular senescence
would be important.
In summary, the results of our investigation on the role of survivin in prostate
cancer progression using a double conditional Pten and Survivin mouse model is
particularly significant because it led to insights of the direct impact of Survivin deletion
occurring simultaneously with that of Pten deletion in the process of tumorigenesis.
Evidence is obtained to link a supporting role of survivin in the progression of PIN
lesions to adenocarcinoma of the prostate in the model system. Additionally, it is
apparent that lesions formed in the absence of survivin are variant in microscopic
phenotypes with hallmarks of hypertrophy, exfoliation and scenescence. These findings
offer a great value for future pre-clinical or clinical investigation for the control of PIN
lesions. It is projected from the findings of this study that inhibition of survivin activity in

84

the human patients may retard or block progression of the PIN lesions, and, thereby
extending the therapeutic window for prostate cancer.

85

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89

CHAPTER FOUR: CONCLUSIONS AND REMARKS ABOUT FUTURE
STUDIES

4.1 Conclusions
Our exploration of the prostate cancer stem cell world led to important and
interesting novel insights. First, we showed that a subpopulation of cells within the
primary and castration-resistant prostate tumor of our conditional Pten deletion mice,
having a high level of expression of Sca-1 and CD49f (LSC
hi
) possesses both self
renewal and the potential to differentiate into basal and luminal cell types. These
particular stem cell-like characteristics were assessed by the ability of these cells to
form spheroids in culture over multiple generations as well as their capacity to generate
tumor-like glandular structures in vivo. The identification of multiple cell types (basal,
transit-amplifying, and luminal) residing within the spheroids in vitro and the glandular
structures in vivo confirmed LSChi cells’ differentiation potential. Second, for the first
time, the study revealed the importance of the cancer associated fibroblasts’ (CAF)
influence on the putative cancer stem cell (CSC) population. Androgen-dependent
(AD)-CAFs were shown to significantly increase the spheroid forming efficiency of AD-
cancer (AD-Ca) CSCs in vitro compared to urogenital sinus mesenchyme (UGSM) or
normal prostate fibroblasts (NPFs). In vivo glandular structures generated by AD-Ca
CSCs in the presence of AD-CAFs also exhibited larger and more complex tumor-like
morphology and were higher in number compared to those formed in the presence of
UGSM or NPFs. There also seems to be a difference in the characteristics of AD-Ca
CSCs and castrate-resistant prostate cancer (CRPC) CSCs in their response to the

90

exposure to AD-CAFs or castrate-resistant (CR)-CAFs. The CRPC CSCs exhibited
preferential response to CR-CAFs in terms of their spheroid-forming and glandular
structure-forming potentials, suggesting a possible co-evolution of the tumor epithelial
and stromal compartments. Finally, real-time PCR analysis on the expression patterns
of AD-Ca CSCs led to the elucidation of a signature expression profile of Sca-1 (high),
CD49f (high), CK5 (high), p63 (high), Survivin (high), RUNX2 (high), CD44 (low),
CD133 (low), CK18 (low), and Androgen Receptor (low). It is now important to assess
the expression of various cellular markers including those listed above on the CRPC
CSCs as a means to detect the potential molecular differences between the two
populations of CSCs. Similarly, it will be critically important to discern the profile of
factors secreted by the CAFs in relation to their origin from AD or CRPC cancers for the
clues on co-evolution of the stroma.
Our investigation of the role of survivin in prostate cancer progression has also
resulted in a number of noteworthy findings. First, we demonstrated that simultaneous
deletion of Survivin and Pten in our mouse model impedes the prostate lesions from
advancing to adenocarcinoma. Examining mouse groups at ages 8, 20, 36, and 52
weeks, we found only PIN lesions in conditional Pten deleted mice as old as 52 weeks
that lacked both Survivin alleles, whereas both conditional Pten knockout mice with
intact or monoallelic inactivation of Survivin manifested adenocarcinoma by this time
point and exhibited early carcinoma as early as 8 weeks and 20 weeks, respectively.
Development of carcinoma in cPten
-/-
S
-/-
mice at older ages remains to be investigated.
Second, the PIN lesions observed in cPten
-/-
S
-/-
mice were strikingly atypical, displaying
many hypertrophic cells, polyploidy, and cellular exfoliation. This type of PINs was

91

rarely observed in the other two groups of mice, cPten
-/-
S
+/+
and cPten
-/-
S
+/-
. Third, mice
lacking Pten and both Survivin alleles exhibited enhanced apoptosis (indicated by
histological analysis, increased expression of cleaved caspase-3 and down-regulation
of the activated form of the pro-survival factor Bcl-2) and decreased proliferation index
(assessed by Ki67 expression level) compared to the other two groups of conditional
Pten deletion mice, in line with previous reports on survivin’s functions as an inhibitor of
apoptosis and a regulator of cell division in normal and cancer cells. Expression level of
XIAP, another inhibitor of apoptosis family member, was also found to decrease with
Survivin homozygous deletion, pointing to the possible interaction of the two proteins
that has also been suggested by other studies (1, 2). Lastly, this study also led to some
novel observations that deserve more attention in future work. One such point is the
possible association of survivin and cellular senescence, which was detected at all ages
of cPten
-/-
S
-/-
mice, but only in younger ages in cPten
-/-
S
+/+
and cPten
-/-
S
+/-
mice.
Another notable insight was the observation that p63 expression level seems to be
correlated with survivin level in the tissue, indicating the possible role of survivin in
cancer stem cells, as suggested by our previous findings of survivin and Runx2 up-
regulation in the putative prostate cancer stem cell compartment.
Overall, our studies have contributed some important findings to facilitate the
progress of several aspects of prostate cancer research. We provided clues of the
existence of a rare cancer stem cell population, showing enhanced self-renewal and
differentiation abilities in the presence of cancer-associated fibroblasts from its
microenvironment, which may be an underlying mechanism in prostate recurrence. We
also demonstrated the strong role of survivin in promoting prostate cancer progression

92

to adenocarcinoma state. We hope that together, these results can lead to a better
understanding of prostate cancer and inspire future studies that may lead to more
efficient therapy against the disease.
4.2 Future studies
In the course of our investigation on prostate cancer stem cells, new questions
arise. An important point to address is where the prostate cancer stem cell population
is derived from. Did normal stem cells residing in the prostate undergoing mutations
give rise to cancer stem cells that becomes the foundation for the bulk of the malignant
tumors? Did cancer stem cell population originate from differentiated luminal cells that
acquired mutations leading to malignancy and the development stem cell-like
characteristics to facilitate their growth and survival? Our study indicates that there is a
difference between AD-CSCs and CRPC CSCs in their response to AD-CAFs and
CRPC-CAFs, as well as the type of glandular structures they generate. It is therefore
crucial to identify distinguishing characteristics of CRPC CSCs over AD-Ca CSCs that
may be responsible for their ability to thrive in the low-androgen state and develop
recurrent prostate tumors after androgen ablation. Since normal prostate stem cells
were suggested not to depend on the presence of androgen for their functions (3), it is
likely that AD-CSCs already have the ability to survive and proliferate in androgen-
depleted state. It is possible that AD-CSCs, in an environment with normal androgen
level, do not naturally have the ability to survive in low androgen levels, but can adapt to
do so when exposed to that state by altering their molecular characteristics. Should this
be the case, it is important to investigate whether all of the AD-CSCs possess this

93

potential or only a fraction that would undergo selection in the presence of low
androgen. Currently we are able to enrich a putative CSC population using the same
markers used to isolate normal prostate stem cells. Development of distinct surface
markers that differentiate CSCs from normal prostate stem cells would then be of great
value in coming up with new therapeutic strategies that would target the CSC population
specifically. Our work on prostate cancer stem cells also sheds light on the importance
of the CAFs in enhancing the CSCs stemness and differentiation potential that may
facilitate cancer progression. It would then be beneficial to decipher what molecules
and proteins are expressed and secreted by the CAFs that serve to support various
functions of CSCs and distinguish the underlying differences between AD-CAFs and
CRPC CAFs.
Even though survivin is already an extensively-studied protein due to its
promising therapeutic potentials, more studies on the protein still need to be done. In
our investigation that suggests the strong role of survivin in prostate cancer progression,
we noted some interesting observations that bring into focus some additional less-
known possible functions of survivin. The finding that Survivin homozygous deletion
resulted in more pronounced senescence within the prostate tissue compared to intact
and single allele inactivation of Survivin suggests that survivin may function to suppress
cellular senescence that is found to be implicated in early cellular transformation as a
response to halt the course of tumor progression. Whether this is a direct or secondary
effect of survivin remains to be deciphered. Another interesting possible function of
survivin to explore is its role in regulating various aspects of cancer stem cells. This is
brought about by the observation that p63 expression level seemed to decrease in the

94

absence of survivin. p63 is a prominent marker of basal cells, where stem cell
population is thought to reside. The initial finding of a possible correlation between p63
and survivin level, supported by our earlier detection of high survivin expression in the
putative cancer stem cells, suggest a novel role that survivin may play in promoting
prostate cancer progression. The results of our study also suggest the potential of
using the conditional Pten dele tion mouse model for pre-clinical studies to test various
anti-survivin therapeutic modalities in conjunction with other apoptotic inducers to halt
the progression of prostate cancer.

95

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105

APPENDIX: ABBREVIATIONS
AD – Androgen dependent
AD-Ca – Androgen-dependent cancer
AIP – Aryl hydrocarbon receptor-interacting protein
AR – Androgen receptor
BLI – Bioluminescence imaging
BMP – Bone morphogenetic protein
CAFs – Cancer-associated fibroblasts
CK8 – Cytokeratin 8
CR – Castrate-resistant
CRPC – Castrate-resistant prostate cancer
CSCs – Cancer stem cells
HSP – Heat shock protein
IAP – Inhibitor of apoptosis
LS – Lin
-
Sca-1
+

LSC – Lin
-
Sca-1
+
CD49f
+

LSC
hi
- Lin
-
Sca-1
hi
CD49f
hi
LSC
me
- Lin
-
Sca-
me
CD49f
me
mPIN – Murine prostatic intraepithelial neoplasm
N- Normal
NPFs – Normal prostate fibroblasts
P-Akt – Phosphorylated Akt
PB - Probasin

106

APPENDIX: ABBREVIATIONS (C ONT’D )
PIN – Prostatic intraepithelial neoplasm
PTEN – Phosphatase and tensin homolog deleted on chromosome 10
T - Tumor
UGSM – Urogenital sinus mesenchyme

Abstract (if available)

Abstract Our study of prostate cancer is centered on a conditional mouse model based on the Cre/lox recombination technology to inactivate the tumor suppressor gene Pten, whose function is frequently lost in human prostate cancer. This model proved to be a powerful tool in our investigation on the presence and characteristics of putative cancer stem cell population and its interaction with the tumor microenvironment, as well as on the role of survivin, a cancer-specific anti-apoptotic protein in prostate cancer progression. The combination of conditional Pten deletion and luciferase-expressing mouse model allowed us to non-invasively follow the tumor growth through bioluminescence imaging, permitting the collection of tumors at either the androgen-dependent (AD) primary growth phase or the recurrent phase when castration-resistant prostate cancer (CRPC) is formed after initial regression from androgen-deprivation therapy. Utilizing cell surface markers shown to enrich normal prostate epithelial stem cells (Lin⁻Sca-1⁺CD49f⁺), we were able to further restrict the parameters of selection to enrich putative cancer stem cells (CSCs) from both androgen-dependent and castrate-resistant prostate tumors. These populations of cells exhibited the ability to self-renew and differentiate to multiple cell types in vitro and in vivo, in concordance to known characteristics of normal prostate tissue stem cells. Along with the detection of expression of certain expected stem cell-like markers, evidence was obtained that the CSCs from the model also retain a high level of survivin expression like the cancer cells. We demonstrated the contribution of the cancer-associated fibroblasts (CAFs) of the tumor microenvironment in enhancing the putative cancer stem cells’ self-renewal and differentiation potentials. We also found that in vivo growth and differentiation of CSCs from CRPC cancer were better supported by CAFs derived from CRPC cancer compared to those from AD cancer. These results suggested that signaling proteins that are secreted by stage-specific CAFs might support and potentiate the stemness and tumorigenic properties of the corresponding CSCs. This novel finding deserves to be further investigated, particularly since stromal fibroblasts remain as an understudied cellular compartment of the prostate cancer. ❧ Our examination of the role of survivin in prostate cancer was achieved through the generation of a prostate epithelium-specific double knock-out mouse model lacking Pten and Survivin. With Survivin deletion alone, normal prostate organogenesis and growth as well as fertility of mice were not found to be impaired. Survivin homozygous deletion in conditional Pten knockout mice, however, resulted in the delay of prostate cancer progression in the sense that even up to 52 weeks of observation, no adenocarcinoma but only premalignant lesions, namely prostatic intraepithelial neoplasms (PINs) were detected. This was in contrast to adenocarcinoma formation by 36 weeks in many animals of the control tumor group or the heterozygous Survivin deletion group. Prostate tumors lacking survivin appeared to display enhanced apoptosis, decreased proliferation index, increased senescence, and a high degree of hypertrophic cells, some of these characteristics being atypical of the usual PIN lesions. Our findings from this direct in vivo genetic study demonstrate the importance of survivin in prostate cancer progression potentially via its anti-apoptotic and cell division regulatory roles, as supported by previous studies, as well as open up possibilities of survivin’s association with cellular senescence and cancer stem cells that remains to be explored in the future.

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Creator Adisetiyo, Helty Adisetiyo (author)

Core Title Exploration of the roles of cancer stem cells and survivin in the pathogenesis and progression of prostate cancer

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Genetic, Molecular and Cellular Biology

Publication Date 09/13/2012

Defense Date 08/30/2012

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

Tag androgen-dependent prostate cancer,cancer associated fibroblasts,cancer stem cells,castrate-resistant prostate cancer,OAI-PMH Harvest,prostate cancer,survivin,tumor microenvironment

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Roy-Burman, Pradip (committee chair), Frenkel, Baruch (committee member), Hong, Young-Kwon (committee member)

Creator Email adisetiy@usc.edu,heltya@yahoo.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-95860

Unique identifier UC11289175

Identifier usctheses-c3-95860 (legacy record id)

Legacy Identifier etd-AdisetiyoH-1195.pdf

Dmrecord 95860

Document Type Dissertation

Rights Adisetiyo, Helty Adisetiyo

Type texts

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

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA