1. health and fitness
2. disease
3. cancer

THE EXPRESSION, REGULATION AND
FUNCTION OF KALLIKREIN 4
IN PROSTATE CANCER
Rachael Collard
B.AppSc (Hons)
Centre for Molecular Biotechnology
School of Life Sciences, QUT
A thesis submitted to the Queensland University of Technology
for the degree of Doctor of Philosophy
2003
KEYWORDS
Kallikrein, prostate cancer, metastasis, KLK4/hK4, hormonal regulation, androgen,
migration, invasion, attachment.
i
ABSTRACT
Prostate cancer is a significant health problem faced by aging men. Currently, diagnostic
strategies for the detection of prostate cancer are either unreliable, yielding high
numbers of false positive results, or too invasive to be used widely as screening tests.
Furthermore, the current therapeutic strategies for the treatment of the disease carry
considerable side effects. Although organ confined prostate cancer can be curable, most
detectable clinical symptoms occur in advanced disease when primary tumour cells have
metastasised to distant sites - usually lymph nodes and bone.
Many growth factors and steroids assist the continued growth and maintenance of
prostatic tumour cells. Of these mitogens, androgens are important in the development
of the normal prostate but are also required to sustain the growth of prostate cancer cells
in the early stage of the disease. Not only are androgens required in the early stage of
disease, but also many other growth factors and hormones interact to cause uncontrolled
proliferation of malignant cells.
The early, androgen sensitive phase of disease is
followed by an androgen insensitive phase, whereby androgens are no longer required to
stimulate the growth of the tumour cells. Growth factors such as transforming growth
factor α and β (TGFα/β), epidermal growth factor (EGF), basic fibroblast growth factor
(bFGF), insulin-like growth factors (IGFs), Vitamin D and thyroid hormone have been
suggested to be important at this stage of disease. Interestingly, some of the kallikrein
family of genes, including prostate specific antigen (PSA), the current serum diagnostic
marker for prostate cancer, are regulated by androgens and many of the aforementioned
growth factors.
The kallikrein gene family is a group of serine proteases that are involved in a diverse
range of physiological processes: regulation of local blood flow, angiogenesis, tissue
invasion and mitogenesis. The earliest members of the kallikrein gene family (KLK1KLK3) have been strongly associated with general disease states, such as hypertension,
inflammation, pancreatitis and renal disease, but are also linked to various cancers.
Recently, this family was extended to include 15 genes (KLK1-15). Several newer
ii
members of the kallikrein family have been implicated in the carcinogenesis and tumour
metastasis of hormone-dependent cancers such as prostate, breast, endometrial and
ovarian cancer.
The aims of this project were to investigate the expression of the newly identified
kallikrein, KLK4, in benign and malignant prostate tissues, and prostate cancer cell lines.
This thesis has demonstrated the elevated expression of KLK4 mRNA transcripts in
malignant prostate tissue compared to benign prostates. Additionally, expression of the
full length KLK4 transcript was detected in the androgen dependent prostate cancer cell
line, LNCaP.
Based on the above finding, the LNCaP cell line was chosen to assess the potential
regulation of full length KLK4 by androgen, thyroid hormone and epidermal growth
factor.
KLK4 mRNA and protein was found to be up-regulated by androgen and a
combination of androgen and thyroid hormone. Thyroid hormone alone produced no
significant change in KLK4 mRNA or protein over the control. Epidermal growth factor
treatment also resulted in elevated expression levels of KLK4 mRNA and protein.
To assess the potential functional role(s) of KLK4/hK4 in processes associated with
tumour progression, full length KLK4 was transfected into PC-3 cells - a prostate cancer
cell line originally derived from a secondary bone lesion.
The KLK4/hK4 over-
expressing cells were assessed for their proliferation, migration, invasion and attachment
properties.
The KLK4 over-expressing clones exhibited a marked change in morphology, indicative
of a more aggressive phenotype. The KLK4 clones were irregularly shaped with
compromised adhesion to the growth surface. In contrast, the control cell lines (parent
PC-3 and empty vector clones) retained a rounded morphology with obvious cell to cell
adhesion, as well as significant adhesion to their growth surface.
The KLK4 clones
exhibited significantly greater attachment to Collagen I and IV than native PC-3s and
empty vector controls. Over a 12 hour period, in comparison to the control cells, the
KLK4 clones displayed an increase in migration towards PC-3 native conditioned media,
iii
a 3 fold increase towards conditioned media from an osteoblastic cell line (Saos-2) and
no change in migration towards conditioned media from neonatal foreskin fibroblast
cells or 20% foetal bovine serum. Furthermore, the increase in migration exhibited by
the KLK4 clones was partially blocked by the serine protease inhibitor, aprotinin.
The data presented in this thesis suggests that KLK4/hK4 is important in prostate
carcinogenesis due to its over-expression in malignant prostate tissues, its regulation by
hormones and growth factors associated with prostate disease and the functional
consequences of over-expression of KLK4/hK4 in the PC-3 cell line. These results
indicate that KLK4/hK4 may play an important role in tumour invasion and bone
metastasis via increased attachment to the bone matrix protein, Collagen I, and enhanced
migration due to soluble factors produced by osteoblast cells. This suggestion is further
supported by the morphological changes displayed by the KLK4 over-expressing cells.
Overall, this data suggests that KLK4/hK4 should be further studied to more fully
investigate the potential value of KLK4/hK4 as a diagnostic/prognostic biomarker or in
therapeutic applications.
iv
TABLE OF CONTENTS
Keywords
i
Abstract
ii
Table of contents
v
List of Figures
xi
List of Tables
xiv
Abbreviations
xv
Statement of Original Authorship
xviii
Acknowledgements
xix
Chapter 1: INTRODUCTION AND LITERATURE REVIEW
1
1.0
Introduction
2
1.1
The Prostate
3
1.1.1
Prostatic Structure and Function
3
1.2
Diseases of the Prostate
5
1.2.1 Benign Prostatic Hyperplasia (BPH)
5
1.2.2
Prostatic Intraepithelial Neoplasia (PIN)
6
1.2.3
Prostate Cancer
7
1.2.3.1
Diagnosis
9
1.2.3.2
Clinical Considerations
10
1.3
Hormonal and Growth Factor Involvement in the Progression of
12
Prostate Cancer
1.3.1
Androgens
13
1.3.2
Estrogens
15
1.3.3
Vitamin D
15
1.3.4
Thyroid Hormone
16
1.3.5
TGFβ
16
1.3.6
EGF and TGFα
17
1.3.7
IGFs
17
1.3.8
FGF
18
v
1.3.9
VEGF
19
1.4
Prostate Cancer Cell Models
19
1.5
Proteases Associated with Prostate Cancer
1.5.1
Matrix Metalloproteases
21
1.5.2
Urokinase-type Plasminogen Activator
23
1.5.3
The Human Kallikrein Family
23
1.5.3.1
KLK2 and KLK3
27
1.5.3.2
Human KLK4
29
1.5.3.2.1
Expression
29
1.5.3.2.2
KLK4 mRNA Variant Transcripts
31
1.5.3.2.3
Regulation
34
1.5.3.2.4
Functional Studies
35
1.6
Conclusion and Relevance to Project
Chapter 2: MATERIALS AND METHODS
36
40
2.0
Introduction
41
2.1
Materials and Methods
41
2.1.1
Cell Culture
41
2.1.1.1
Resuscitation of Cells from Liquid Nitrogen
41
2.1.1.2
Routine Passaging of Cells
42
2.1.1.3
Preparation of Cryo-Preserved Stocks
42
2.1.2
RNA Extraction
42
2.1.3
Polymerase Chain Reaction (PCR)
43
2.1.3.1
Reverse Transcription-Polymerase Chain Reaction (RT- 43
PCR)
2.1.3.2
Quantitative RT-PCR
44
2.1.4
Gel Purification
45
2.1.5
DNA Sequencing
45
2.1.6
Western Blot Analysis
46
2.1.6.1
46
Intracellular Protein Extraction
vi
2.1.6.2
Protein Quantitation
46
2.1.6.3
Sodium Dodecyl Sulphate-Polyacrylamide Gel
46
Electrophoresis (SDS-PAGE)
2.1.6.4
2.1.7
Western Blotting
47
Immunofluorescence
48
Chapter 3: THE EXPRESSION OF KLK4 IN PROSTATE CANCER
49
3.0
Introduction
50
3.1
Materials and Methods
52
3.1.1
Prostate Tissue Samples
52
3.1.2
RNA Extraction and Conventional RT-PCR
52
3.1.3
Real-Time Quantitative PCR of KLK4 mRNA Transcript Expression
55
3.1.4
Pearson’s Correlation Analysis of Prostate Cancer Specimens
56
3.1.5
Protein extraction
56
3.1.6
Western Blot
57
3.1.7
Immunofluorescence
57
3.2
Results
59
3.2.1 RT-PCR Expression of KLK4 mRNA Transcripts Compared to PSA in 59
Benign and Malignant Prostate Tissues
3.2.2
Real-Time PCR Analysis of KLK4 and PSA mRNA Transcripts in 61
Prostate Cancer and BPH
3.2.3
Correlation of Tumour Grade (Gleason Score) to Transcript Level
3.2.4 RT-PCR Expression of KLK4 and PSA mRNA Transcripts in Prostate
63
63
Cancer Cell Lines
3.2.5
The Expression of hK4 in Prostate Cancer Cell Lines
66
3.2.6
Immunofluorescence
68
3.3
Discussion
71
vii
Chapter 4: THE REGULATION OF KLK4 IN THE
77
PROSTATE CANCER CELL LINE, LNCAP
4.0
Introduction
78
4.1
Materials and Methods
81
4.1.1 Cell Culture
81
4.1.2
PSA Assay
81
4.1.3
RNA Extraction and RT-PCR
82
4.1.4
Real-Time Quantitative PCR of KLK4 mRNA Transcript Expression
82
4.1.5
Protein Extraction
82
4.1.6
Western Blot
83
4.1.7
Quantitation of Signal Intensity
83
4.2
Results
84
4.2.1
Regulation of PSA and KLK4 in LNCaP Cells by DHT and T3
84
4.2.1.1
PSA Assay of Conditioned Medium
84
4.2.1.2
PSA and KLK4 mRNA Regulation by DHT and T3
84
4.2.1.3
PSA and hK4 Protein Expression in Response to DHT and 87
T3 Treatment
4.2.2
Regulation of PSA and KLK4 in LNCaP Cells by EGF
90
4.2.2.1
PSA Assay of Conditioned Medium
90
4.2.1.2
PSA and KLK4 mRNA Regulation by EGF
90
4.2.1.3
PSA and hK4 Protein Expression in Response to EGF
93
Treatment
4.3
95
Discussion
Chapter 5: THE ESTABLISHMENT OF STABLY TRANSFECTED PC-3
102
PROSTATE CANCER CELLS OVER-EXPRESSING FULL LENGTH KLK4
5.0
Introduction
103
5.1
Materials and Methods
105
viii
5.1.1
KLK4 Construct and Mammalian Expression Vectors
105
5.1.2
Lipid-Mediated Transfection
105
5.1.3
Generation of Stably Transfected Clones
107
5.1.4
Confirmation of Stably Transfected Clones
109
5.1.4.1
Collection of Cell Pellets and Conditioned Media
109
5.1.4.2
RT-PCR
109
5.1.4.3
Quantitative RT-PCR
110
5.1.4.4
Western Blotting
110
5.1.4.5
Immunofluorescence
111
5.1.5 Morphological Analysis
111
5.2
Results
112
5.2.1
Generation of Stably Transfected Clones
112
5.2.2 RT-PCR Expression of KLK4 in Transfected Clones
112
5.2.3 Quantitative RT-PCR Expression of KLK4 in Transfected Clones
112
5.2.4
Protein Analysis of hK4 in Transfected Clones
114
5.2.5
Immunofluorescence
117
5.2.6 Cell Morphology
117
5.3
122
Discussion
Chapter 6: FUNCTIONAL CHARACTERISATION OF HK4
126
OVER-EXPRESSING PC-3 CELLS
6.0
Introduction
127
6.1
Materials and Methods
129
6.1.1
Cell Culture
129
6.1.2
Functional Analysis of hK4 Stably Expressing Clones
129
6.1.2.1
MTT Tetrazolium Proliferation Assay
129
6.1.2.2
Preparation of Chemo-Attractants
130
6.1.2.3
Migration (Chemotaxis) Assay
131
6.1.2.4
Blocking Assay
133
6.1.2.4.1
134
hK4 Antibody Blocking Assay
ix
6.1.2.4.2
6.2
Aprotinin Blocking Assay
134
6.1.2.5
Chemo-Invasion Assay
134
6.1.2.6
Attachment Assay
135
138
Results
6.2.1 Effect of hK4 Over-Expressing Stably Transfected Cell Lines on the
138
Rate of Proliferation
6.2.2 Effect of hK4 Over-Expressing Stably Transfected Cell Lines on Cell
140
Motility
6.2.3
Effect of hK4 antibodies on hK4 Mediated Cell Motility
144
6.2.4
Effect of Aprotinin on hK4 Mediated Cell Motility
144
6.2.5 Effect of hK4 Over-Expressing Stably Transfected Cell Lines on Cell
147
Invasion
6.2.6 Effect of hK4 Over-Expressing Stably Transfected Cell Lines on Cell
149
Attachment to Extracellular Matrix Molecules
6.3
152
Discussion
Chapter 7: GENERAL DISCUSSION
160
7.0
Introduction
161
7.1
KLK4 Transcripts in Prostate Tissue Samples and Prostate Cancer Cell
161
Lines
7.2
Regulation of KLK4 Transcripts by Hormones and Growth Factors
165
7.3
Functional Effects of hK4 Over-Expression
167
7.4
Conclusion
174
Chapter 8: REFERENCES
x
175
LIST OF FIGURES
Figure 1.1
Anatomical zones of the prostate gland
5
Figure 1.2
Different stages of prostate tumour development and
8
progression
Figure 1.3
Cellular events leading to metastasis
22
Figure 1.4
The human kallikrein locus
25
Figure 1.5
Schematic diagram of full length KLK4 and alternatively
32
spliced mRNA transcripts.
Figure 1.6
Schematic diagram of full length and variant hK4 proteins
33
Figure 3.1
Location of RT-PCR primers for full length KLK4 and
54
alternatively spliced mRNA transcripts
Figure 3.2
Position of antibodies for full length and variant hK4 proteins
58
Figure 3.3
KLK4 and PSA mRNA transcript expression profile in prostate
60
cancer and BPH patient tissues
Figure 3.4
Real-time PCR analysis of KLK4 mRNA in prostate cancer and 62
BPH
Figure 3.5
Correlation of tumour grade to transcript type
64
Figure 3.6
KLK4 and PSA mRNA transcript expression profile in prostate
65
cell lines
Figure 3.7
hK4 and PSA protein expression profile in prostate cell lines
67
Figure 3.8
Immunofluorescence analysis for hK4 expression in prostate
69
cell lines using the C terminus Ab
Figure 3.9
Immunofluorescence analysis for hK4 expression in prostate
70
cell lines using the N terminus Ab
Figure 4.1
The regulation of secreted PSA protein by DHT and T3 in
85
LNCaP cells
Figure 4.2
The regulation of PSA and KLK4 mRNA by DHT and T3
Figure 4.3
The expression of hK4 protein in response to DHT and T3
86
treatment
88
Figure 4.4
The regulation of PSA protein by DHT and T3
89
Figure 4.5
PSA protein secretion in response to EGF treatment
91
xi
Figure 4.6
The regulation of PSA and KLK4 mRNA by EGF
92
Figure 4.7
The expression of hK4 protein in response to EGF treatment
94
Figure 5.1
Vector schematics and complete amino acid sequence of hK4
106
expression construct
Figure 5.2
Lipid-mediated transfection protocol
108
Figure 5.3
RT-PCR analysis of full length KLK4 expression in transfected 113
clones
Figure 5.4
Quantitative RT-PCR analysis of KLK4 expression in
115
transfected PC-3 cells
Figure 5.5
Western blot analysis of K4 over-expressing clones and control
116
cells
Figure 5.6
Immunofluorescence analysis for hK4 expression in KLK4
118
transfected PC-3 cells with the N terminus anti-hK4 peptide
antibody
Figure 5.7
Immunofluorescence analysis for hK4 expression in KLK4
119
transfected PC-3 cells with the C terminus anti-hK4 peptide
antibody
Figure 5.8
Cellular morphology of KLK4 transfected PC-3 cells
120
Figure 6.1
Migration assay
132
Figure 6.2.
Rate of proliferation assessed using the MTT tetrazolium
139
proliferation assay
Figure 6.3
Migratory potential for hK4 clones at various time points
141
Figure 6.4
Migratory potential of hK4 clones towards various chemo-
142
attractants
Figure 6.5
Effect of hK4 antibodies on hK4 mediated cell motility
145
Figure 6.6
Effect of aprotinin on hK4 mediated cell motility
146
Figure 6.7
Invasive potential for hK4 over-expressing clones compared to
148
controls
Figure 6.8
Percent attachment of hK4 over-expressing clones and control
cell lines to the extracellular matrix molecules Fibronectin,
Collagen IV and Collagen I
xii
150
Figure 6.9
Potential Mechanisms of hK4 Action
xiii
158
LIST OF TABLES
Table 1.1
Role of growth factors in prostate cancer and metastasis
14
Table 3.1
Surgical and pathology information for tissue preparations
53
from 24 prostate cancer patients and 28 BPH patients
Table 3.2
Oligonucleotide Primers for conventional RT-PCR
55
Table 3.3
Oligonucleotide Primers for Real Time Quantitative RT-PCR
55
xiv
ABBREVIATIONS
µg
microgram
µl
microlitre
µmol
micromole/L
AR
androgen receptor
ARE
androgen response element
ANOVA
analysis of variance
BCA
bicinchoninic acid method
bp
base pairs
BPH
benign prostatic hyperplasia
BSA
bovine serum albumin
cDNA
complementary DNA
DEPC
diethylpyrocarbonate
DMSO
dimethylsulfoxide
DNA
deoxyribonucleic acid
dNTP
deoxynucleotide triphosphate
DRE
digital rectal examination
DTT
dithiothreitol
ECM
extracellular matrix
EDTA
ethylene diamine tetra acetate
EGF
epidermal growth factor
EMSP1
enamel matrix serine protease 1
EMT
epithelial-mesenchymal transition
FGF
fibroblast growth factor
g
grams
HGPIN
high grade prostatic intraepithelial neoplasia
hK(1-15)
kallikrein protein
h
hour
IGF
insulin-like growth factor
kb
kilobase pairs
xv
KDa
kilodalton
KLK(1-15)
kallikrein gene
LB
Luria Bertoni
LCM
laser capture microdissection
M
mol/L
Mg
magnesium
ml
milliliter
MMP
matrix metalloprotease
mRNA
messenger RNA
NaCl
sodium chloride
NaOH
sodium hydroxide
NFF
normal foreskin fibroblasts
ng
nanograms
nmol
nanomole
OD
optical density
PAP
prostatic acid phosphatase
PAR
protease-activated receptor
PIN
prostatic intrepithelial neoplasia
PBS
phosphate-buffered saline
PCR
polymerase chain reaction
pmol
picomole
PSA
prostate specific antigen
RNA
ribonucleic acid
rpm
revolutions per minute
RT-PCR
reverse transcriptase polymerase chain reaction
SDS
sodium dodecyl sulfate
SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
SSC
sodium chloride, sodium citrate
T3
triiodothyronine
TBE
tris-borate, EDTA
TBS
tris-buffered saline
xvi
TGF
transforming growth factor
TIMP
tissue inhibitors of metalloproteases
U
units
uPA
urokinase plasminogen activator
UV
ultraviolet
VEGF
vascular endothelial growth factor
v/v
volume per volume
v/w
volume per weight
xvii
STATEMENT OF ORIGINAL AUTHORSHIP
The work contained in this thesis has not been previously submitted for a degree or
diploma at any other higher education institution. To the best of my knowledge and
belief, the thesis contains no material previously published or written by another person
except where due reference is made.
Signed:
Date:
xviii
ACKNOWLEDGEMENTS
I would like to thank my supervisors Professor Judith Clements and Professor Adrian
Herington for their constant guidance, support, encouragement and friendship. I also
wish to thank Judith for her generosity in the form of the Hormone-Dependent Cancer
Research Scholarship extension which I received.
Dr Tara Veveris-Lowe, my mentor and friend, who has been the best role-model through
my entire PhD, and taught me everything I needed to know and more.
My closest friend, Miss Carolyn Chan, who has been a constant source of inspiration to
me. For her infinite support and encouragement; her friendship and laughter, and for
being the happiest part of my PhD.
Dr Daniel McCulloch who has helped me in so many ways. For his help and advice on a
day to day basis, his friendship and encouragement, and for being my lunch partner after
Carolyn left.
My support network at QUT who have made the PhD journey so much more enjoyable.
I thank them for their advice, friendship, strength and support: Dr Lisa Chopin, Penny
Jeffery, Steve Myers, Nicole Willemsen, Judy Craft, Steve Liew, Lisa Hayes and Eliza
Whiteside.
There are many people to thank for their valuable assistance with aspects of this study:
Mr Greg Ward at the PA Hospital, School of Life Science office staff and CMB support
staff.
The Queensland Cancer Fund, for providing me with a three year PhD
scholarship, travel funding and a part-time job.
Endless thanks to my wonderful parents, family and friends for their love and support
and for giving me a reason to finish what I’d started.
xix
CHAPTER ONE
INTRODUCTION AND LITERATURE REVIEW
Chapter 1
1.0 INTRODUCTION
Prostate cancer is a disease that affects mostly older males and is the second most
common cause of cancer death in the Western world (Jemal et al., 2002). The principal
cause of death from prostate cancer is due to secondary deposits or metastases,
particularly to the skeleton causing debilitating bone pain and pathological fractures.
The primary tumour comprises a population of epithelial cells that have aberrant growth
and differentiation and subsequently proliferate without constraint. Proliferation is
mediated by several factors including androgens (Bentel and Tilley, 1996), insulin-like
growth factor (IGF) ( Iwamura et al., 1993, Sprenger et al., 2001), epidermal growth
factor (EGF) ( Jarrard et al., 1994, Schuurmans et al., 1989) and triiodothyronine (T3)
(Esquenet et al., 1995, Zhang et al., 1999). Initially, the primary tumour remains
localised to the prostate until local invasion occurs via the degradation of the
surrounding extracellular matrix (ECM). The dissolution of the ECM allows malignant
cells to enter the general circulation, leading to the formation of metastatic tumour
deposits. Metastasis is a multifactorial process requiring a range of proteolytic enzymes
that function either directly or indirectly in the degradation of the ECM. These lytic
enzymes include matrix metalloproteases (MMPs), plasminogens and serine proteases
(Vile, 1995).
Tissue kallikreins (KLKs), are members of a multigene family of serine proteases, which
are involved in a wide variety of biological processes including facilitating human
prostate cancer cell proliferation (Sutkowski et al., 1999, Lin et al., 1998a),
and
invasion (Webber et al., 1995, Valdes et al., 2001). Recently, the human kallikrein gene
family has been extended to fifteen members and now includes other kallikrein-like
genes, which are clustered on the same chromosomal locus at 19q.13.4 (Diamandis et
al., 2000b, Harvey et al., 2000, Yousef and Diamandis, 2001). KLK1–3 are expressed in
the prostate to varying degrees (Clements, 1998) and play a role in the proliferative and
invasive processes of cancer progression. KLK3 (Prostate specific antigen - PSA) is
currently the most useful diagnostic marker for prostate cancer (Catalona et al., 1998).
2
Chapter 1
Another kallikrein highly expressed in the prostate, KLK2, that is also associated with
prostate tumours, is highly expressed in poorly differentiated cancer cells (Rittenhouse
et al., 1998) and can be utilised for discrimination of organ from non-organ confined
cancer growth (Haese et al., 2000).
One of the newer kallikreins, KLK4, is also highly expressed in the prostate (Nelson et
al., 1999, Stephenson et al., 1999, Yousef et al., 1999, Hu et al., 2000b, Harvey et al.,
2000, Korkmaz et al, 2001). Like many of the other kallikreins, KLK4 has several
variant mRNA transcripts, which, along with the full length transcript have been shown
to be expressed and regulated in hormone dependent cancers including those of the
endometrium, ovary, breast and prostate. Whilst only a recently described member of
the kallikrein family, an increasing body of evidence suggests that KLK4/hK4 may play
an important role in prostate cancer development and progression due to its ability to
activate and degrade proteins associated with key processes of the tumourigenic
pathway.
This chapter will outline the structure and diseases of the prostate gland and the
hormonal regulation of prostate tumours. Aspects of the human kallikrein gene family
will be briefly outlined, with KLK4 reviewed in detail, with respect to its expression
profile and potential regulation and function in prostate disease.
1.1 THE PROSTATE
1.1.1 Prostatic Structure and Function
The prostate is a solid, pear-shaped, glandular organ situated inferior to the base of the
urinary bladder and is traversed by the anterior portion of the prostatic urethra. Its
posterior surface is penetrated by ejaculatory ducts, which enter the prostatic urethra
(Tortora, 1995). The human prostate gland is a composite organ consisting of several
glandular and nonglandular components, which weighs an average of 60 grams in the
adult male (Lalani et al., 1997). The nonglandular tissue includes the indistinct
fibromuscular capsule and the fibromuscular stroma, in which the numerous glands are
3
Chapter 1
embedded (McNeal et al., 1988). The glands function in producing acid secretions
containing citrate, phosphatase and several proteolytic enzymes, all of which constitute
25% of the volume of semen (Tortora, 1995). The fibromuscular capsule envelopes the
prostate and is composed of an inner layer of smooth muscle and an outer fibrous
connective tissue (Ayala et al., 1989). The prostate is divided into three distinct zones:
the transitional, central and peripheral zones (Figure 1.1).
The transitional zone forms 5% of the glands volume and contains moderately compact
fascicles of smooth muscle and is the site of 10% of prostate cancers. The central zone
comprises 25% of the prostatic volume and surrounds the transitional zone. Unlike the
peripheral and transitional zones, the ducts are large and irregular. Additionally, the
glands are complex with tall columnar, pseudostratified, papillary infoldings.
The
stroma is densest in the central zone, followed by the transitional zone and is least dense
in the peripheral zone (Bostwick & Dundore, 1997).
The peripheral zone is the largest and constitutes 70% of the gland’s volume. The
stromal cells, smooth muscle and fibroblasts, which comprise the connective tissue
framework, maintain the structure of the glandular portion of the prostate. The glandular
component of the prostate is composed of large peripheral ducts. The acini and ducts
contain secretory, basal and neuroendocrine cells. The secretory/glandular epithelial
cells secrete PSA, prostatic acid phosphatase (PAP), acid mucin, other secretory
products in addition to expressing the androgen receptor (Bostwick & Dundore, 1997,
Lalani et al, 1997).
The basal cells of the prostate form a flattened layer of
inconspicuous cells at the periphery of the glands separating the secretory epithelial cells
from the basement membrane and stroma. These cells are thought to act as stem cells
that repopulate the secretory cell layer (Bonkhoff et al., 1994). The basal cells also
display epidermal growth factor receptors suggesting a role in growth regulation,
however they contain little or no PSA, PAP or acid mucin (Maygarden et al., 1992).
The neuroendocrine cells are the least common cell type of the prostatic epithelium.
Their function is unknown, however it has been postulated that they exist to serve an
4
Chapter 1
endocrine-paracrine regulatory
role
in growth
and development, similar to
neuroendocrine cells in other organs (Aprikian et al., 1993, Bonkhoff et al., 1991).
1.2 DISEASES OF THE PROSTATE
1.2.1 Benign Prostatic Hyperplasia
Abnormal growth, either benign or malignant, is common in the prostate gland. Benign
prostatic hyperplasia (BPH) arises from the secretory/glandular epithelial cells that line
the acini and ducts of the prostate gland. Although the pathogenesis of BPH is not well
bladder
C
T
P
Figure 1.1 Anatomical zones of the prostate gland
Key:
T: transitional zone; C: central zone; P: peripheral zone.
modified from Algaba et al., 1996.)
5
(Reproduced and
Chapter 1
understood, there is general agreement that it begins with stromal alterations, which then
stimulate growth and variably alter the differentiation of associated epithelial cells
(McNeal et al., 1988). Interestingly, most prostate cancers arise in prostates that already
have BPH. However, BPH originates in the transitional zone while the peripheral zone is
the most prevalent site for prostate cancer. BPH is easily distinguished from prostate
cancer histologically, as BPH has a distinct basal cell layer and is characterised by an
altered stromal-epithelial arrangement causing an increase in the formation of atypical
epithelial glands, with distinct stromal configurations (Bonkhoff and Remberger, 1996).
BPH is the most common nonmalignant condition to affect men in developed countries
and is the most frequent benign condition found in the prostate, occurring in more than
70% of men aged 70 years or greater (Jonler et al., 1994, Ramsey, 2000). Due to the
anatomical position of the prostate, urethra and bladder, urinary obstruction is the most
common symptom of BPH (Medina et al., 1999). Although BPH is not the premalignant
precursor of prostate cancer (Lalani et al., 1997), this condition has significant
similarities with prostate cancer: both show increased prevalence with age (although
BPH usually occurs 15-20 years earlier), require androgenic stimulation and may
respond to androgen deprivation (Hollander and Diokno, 1996). The current clinical
marker for prostate cancer, PSA, is known to increase with age and is associated with
BPH (Bo et al., 2003). While age-specific reference ranges have been recommended
(Richardson and Oesterling, 1997), subsequent studies have demonstrated that cancer
detection (sensitivity) is significantly higher with percent free PSA than with agespecific total PSA reference ranges (Catalona et al., 2000, Saw and Aw, 2000).
Nevertheless, considerable uncertainty remains when distinguishing between BPH and
prostate cancer using the PSA test (and its variations). This will be discussed later in
this review.
1.2.2 Prostatic Intraepithelial Neoplasia (PIN)
PIN, by definition, is characterised as a neoplastic transformation within the epithelial
cells of the prostate.
PIN is subdivided into low and high grade lesions, with the
distinction between the two based on the degree of architectural and cytological changes.
6
Chapter 1
In low grade PIN, there is proliferation of secretory cells with irregular spacing,
pleomorphic nuclei and an intact basal cell layer. High grade PIN (HGPIN) is typified
by enlarged cells with increased nuclear/cytoplasmic ratio, prominent nucleoli, coarse
chromatin clumping along the nuclear membrane and variable degrees of disruption of
the basal cell layer.
The links that associate HGPIN and prostate cancer are well defined (but a detailed
description is beyond the scope of this chapter). Briefly, it has been shown that the
prevalence of both HGPIN and carcinoma increase steadily with advancing age (Sakr et
al., 1993). HGPIN is more frequent in prostate glands that harbour carcinoma compared
to benign prostates (McNeal and Bostwick, 1986), appears at a younger age in AfricanAmericans, a higher risk racial group for prostate cancer incidence and mortality, and
both lesions occur primarily in the peripheral zone of the prostate (Sakr et al., 1993,
McNeal and Bostwick, 1986). In addition, it is well recognised by urologic pathologists
that areas of transition between HGPIN and cancer are frequently encountered in the
peripheral zone of the gland with microscopic foci in which ducts with HGPIN appear to
be in continuity with smaller, separate malignant acini of prostate carcinoma. This
transition has been used to suggest a progression of prostatic neoplasia from a non
invasive into an invasive form, with HGPIN representing the non-invasive phase
(McNeal et al., 1991). Furthermore, the basal cell layer normally present within benign
ducts and acini is variably interrupted in HGPIN and is absent in adenocarcinoma
(Bostwick et al., 1997) (see Figure 1.2).
1.2.3 Prostate Cancer
Prostate adenocarcinoma is the most common cancer in men and the second most
common cause of cancer death, only falling shortly behind lung cancer. However,
statistics indicate that the lifetime probability for an Australian man to be diagnosed with
prostate cancer is 13% and the probability of dying from it is 3% (Coates and
Armstrong, 1994, Hsing et al., 2000). In men over fifty years of age, cancer foci can be
detected in more than 30% of prostates at the time of autopsy and this figure increases to
nearly 90% in men in their nineties (Holund, 1980). These figures suggest that most
men will die of other causes, as the prevalence of prostate cancer is much greater than
7
Chapter 1
A
B
PIN
D
Gleason Pattern 3
C
Gleason Pattern 1
Gleason Pattern 2
F
E
Gleason Pattern 4
Gleason Pattern 5 + 3
G
Metastatic Cancer
Figure 1.2 Different stages of prostate tumour development and progression
H&E-stained sections of various histological tumour patterns for the most commonly
used grading system for prostate cancer as well as the precursor lesion high grade PIN.
Panel A: micropapillary type of high grade PIN. Panel B shows well-differentiated
carcinoma even sized glands with a back-to-back pattern. Panel C shows Gleason
pattern 2 with relatively even-shaped and -sized glands. Panel D shows the variability in
the size of malignant glands and an in.ltrating pattern, typical features of pattern 3.
Perineural invasion is present in the center. Panel E reveals fused glands with clear
cytoplasm, typical of Gleason pattern 4. Panel F exhibits pattern 5 (right half) and
pattern 3 (left half) of the Gleason pattern. Panel G shows metastatic prostate carcinoma
involving bone, strongly positive for PSA immunoperoxidase stain. Original
magnification x100. Modified and reproduced from (Karan et al., 2003).
8
Chapter 1
the death rate. Yet it is not possible to distinguish between patients whose cancer will
remain clinically latent from those with potentially fatal cancer due to the heterogeneity
of each individual’s tumour with respect to stage and grade (Aihara et al., 1994).
Furthermore, clinical features in metastatic disease also vary between patients. As 70%
of prostate tumours are found in the peripheral zone, a tumour may not produce any
urinary-related symptoms until it reaches an advanced state, due to its distance from the
urethra (Ahmed et al., 1997).
1.2.3.1 Diagnosis
Diagnosis of prostate cancer is based on the suspected asymmetry of the gland detected
by digital rectal examination (DRE), subsequent biopsy and serum PSA levels. DRE is a
long-established test used by physicians to detect palpable changes in the prostate gland
but it can only detect cancers that are relatively large and the majority of cancers occur
in regions that are not accessible by DRE (Selley et al., 1997). The abnormal cellular
growth disrupts the prostatic architecture allowing PSA to be released into the
circulation at high concentrations, providing the basis for the PSA serum test (Lalani et
al., 1997; Barry, 2001).
Histological grade of biopsy tissue is assigned using the Gleason grading system
(Gleason, 1966) by observing the architectural patterns or degree of differentiation of the
gland; that is, whether the cells form glands that resemble the normal prostate. A low
grade will be the most differentiated, and scored with a Grade of 1, while a poorly
differentiated, rapidly growing cancer will be assigned a Grade of 5 (see Figure 1.2). As
most patients present with a heterogeneous population of cancer cells, the most common
cancer pattern of cellular structure observed is the primary grade, while the secondary
grade (comprising at least 5% of the cancer) is the second most common pattern.
Consequently, the ‘Gleason score’ is obtained by adding these two values. For example,
a biopsy which shows 60% of Grade 3 tumour and 40% of Grade 4 tumour, has a
Gleason score of 3+4=7. The most common Gleason score is 3+3=6, where virtually all
the tumour is Grade 3 (ie. irregular infiltration of the stroma, but no fusion of glands).
Additionally, ‘staging’ of the cancer represents how advanced the tumour is. Stage A
and B are clinically undetectable cancers confined to the gland, while Stage C tumours
9
Chapter 1
present with extracapsular extension and Stage D is metastatic disease associated with
lymph node involvement and metastasis to bone or visceral organs.
Although PSA is currently the most useful marker for early detection of prostate cancer,
it does not specifically discriminate between prostate cancer and BPH (Becker et al.,
1997; Rosalki & Rutherford, 2000). Furthermore, the PSA test cannot distinguish
between slow growing/latent disease and aggressive/metastatic cancer. Serum PSA can
be elevated in benign prostatic hyperplasia (BPH) and prostatitis, and other nonmalignant forms of prostatic disease, as well as in prostate cancer (Bunting, 1995).
Additionally, serum PSA levels may not always be elevated in cancer suggesting that
there is a need for a more discriminating marker that is both more sensitive and specific
for malignant disease. Recently, various applications of different forms of PSA as
adjunct markers have improved the value of PSA testing (Mikolajczyk and Rittenhouse,
2003, Oremek et al., 2003, Wan et al., 2003, Wesseling et al., 2003). Despite the
availability and use of PSA, and recently the kallikrein, hK2 (Kwiatkowski et al., 1998,
Recker et al., 1998, Partin et al., 1999, Becker et al., 2000, Nam et al., 2000, Scorilas et
al., 2003), as markers for diagnostic and prognostic use in prostate disease management,
additional markers are required to definitively differentiate benign from malignant forms
of prostate disease.
1.2.3.2 Clinical Considerations
Up to 70% of patients with advanced prostatic cancer have bone metastases. Prostate
cancer cells that metastasise to bone typically trigger localised increases in bone
formation by osteoblasts, and these osteoblastic lesions are usually associated with
regions of increased osteolytic activity. Studies have shown that new bone formation is
preceded by local osteolysis and that both increased osteoblastic activity and marked
osteolysis can be seen, with the osteolytic component compromising bone integrity
(Urwin et al., 1985, Clarke et al., 1991, Koutsilieris, 1993, Lange and Vessella, 1998,
Lee et al., 2003). Metastases are found most frequently in lumbar vertebrae, followed
by the sternum, pelvic bones, ribs and femurs (Harada et al., 1992). Bone metastasis is
generally associated with a poor prognosis as the growth rate of the secondary tumour in
10
Chapter 1
bone marrow is considerably greater than that of the slowly growing primary prostatic
tumour (Berrettoni et al., 1986, Keller et al, 2001). Not only does it cause debilitating
bone pain, pathologic fractures, nerve compression syndromes and hypercalcaemia, it
also indicates that the malignant process is incurable (Scher & Chung, 1994).
The
preferential dissemination of prostate cancer cells to bone has been explained by a
number of theories. The mechanical mechanism of retrograde flow of prostate cancer
cells through Batson’s plexus of veins that run between the prostate and the spine has
long been thought to facilitate the process of bone metastasis (Batson, 1995). It has also
been hypothesised that in order to thrive in the bone environment, cancer cells must
acquire “bone cell-like” or osteomimetic properties (Koeneman et al., 1999). The “seed
and soil” theory coined by Paget stresses the importance of the soil, or fertility, of an
organ, which selectively permits metastatic tumour growth in certain organs because of
enhanced adhesion (Nicolson & Winkelhake, 1975), chemotaxis (Hujanene &
Terranova, 1985) or growth (Manishen et al., 1986) at these sites.
Clearly, the
development of new and effective therapeutic treatments for the management of late
stage prostate carcinoma depends therefore on a better understanding of the mechanisms
that underlie the predilection of this malignancy to bone.
The current therapeutic strategies for the treatment of prostate cancer are radical
prostatectomy, radiotherapy and hormone ablation treatment, depending upon the age of
the patient, stage and extent of the disease and other individual factors. Although radical
prostatectomy and radiotherapy are the most common treatments for localised prostate
cancer, both surgery and radiation therapy have considerable side effects including
stricture, bowel injury and significant rates of incontinence, impotence and mortality
(Brawer et al., 2001).
As detailed in the next Section, androgens affect the growth and development of both
normal prostatic cells and the initial growth of prostate cancer cells (Huggins and
Hodges, 1941). Options for androgen blockade which primarily include orchiectomy,
luteinizing hormone-releasing agonists and antagonists, and nonsteroidal antiandrogens
(Oottamasathien and Crawford, 2003) can be successful in early androgen-dependent
disease, resulting in stabilisation or regression in most patients. However, with time
11
Chapter 1
cancer relapse will occur because androgen blockade alone is not curative (Catalona,
1994, Newling, 1997, See, 2003). Recently, it has been suggested that androgen ablation
therapy may select for, or induce, cells with mutational instability, allowing the tumour
to become androgen-independent (Feldman and Feldman, 2001). Even so, the switch
from androgen-sensitive to insensitive cancer is not well understood. In patients with
hormone-refractory/androgen insensitive disease, palliative care is currently the only
treatment available.
In an attempt to understand why tumours progress to an androgen independent
phenotype, numerous studies examining the androgen receptor have been undertaken.
Sequencing of the AR gene from many prostatic tumours and prostate cancer cell lines
show point mutations which produce mutant receptor proteins that are unable to bind
androgens, unable to signal androgen-binding or show reduced response to the ligand
(Veldscholte et al., 1990, Bentel and Tilley, 1996, Zhao et al., 1999, Marcelli et al.,
2000, Buchanan et al., 2001, Thin et al., 2002). Importantly, the AR mutations observed
are only found in metastatic disease and are not detected in individuals with organ
confined disease (Marcelli et al., 2000).
Furthermore, numerous studies have
established that other hormones such as estrogens, progestins, adrenal androgens,
glucocorticoids, cortisol and the non-steroidal antiandrogens, hydroxyflutatmide and
nilutamide, are capable of binding to mutated receptors with greater affinity (Tan et al.,
1997, Veldscholte et al., 1990, Zhao et al., 2000).
1.3 HORMONAL
AND
GROWTH FACTOR INVOLVEMENT
IN THE
PROGRESSION
OF
PROSTATE CANCER
In the prostate, complex interactions occur between peptide growth factors and growth
modulators (including some kallikreins) that may be regulated either by androgen or
independently by other factors. The expression, regulation, and production of many of
these growth factors are modified in prostate cancer.
12
Chapter 1
1.3.1 Androgens
The development and maintenance of normal prostatic structure and function is
dependent upon male steroid hormones, androgens, which exert their effects on stromal
and epithelial cells (Bentel and Tilley, 1996). Upon removal of the androgen supply, the
prostate undergoes atrophy and involution as a direct result of epithelial cell apoptosis
(Montalvo et al., 2000, McConnell, 1990, 1995). Androgens, namely testosterone,
circulate in the blood until, in the prostate, testosterone is converted to
dihydrotestosterone (DHT) by 5α-reductase.
Binding of DHT to the androgen
receptor (AR) causes a conformational change in the AR that exposes DNA binding
sites, which on entering the nucleus of the cell, bind to specific DNA sequences known
as androgen response elements (ARE). AREs are located in the promoter region of all
androgen-regulated genes. In the prostate, androgens increase the transcription of a
number of mitogenic growth factors in epithelial and stromal cells which can act in an
autocrine and/or paracrine manner on the epithelium to regulate cell growth,
differentiation and apoptosis (Ware, 1994, Farnsworth, 1999). Additionally, the
DHT/AR complex up-regulates the transcription of PSA or KLK3, and the closely
related kallikrein, KLK2 ( Riegman et al., 1991, Murtha et al., 1993,).
Although the etiology of prostate cancer is poorly understood, it is known that the
growth and maintenance of cancerous cells in the early stages of disease is dependent
upon androgens. In this initial phase, androgens and variety of other growth factors act
in concert to sustain tumour cell proliferation, however, the cells progress to a stage
where they are no longer responsive to androgenic stimuli (the "hormonal escape"
phase) and growth of the tumour continues independently of androgenic control. Once
the tumour has reached the androgen insensitive phase, it is likely that it will progress to
the metastatic state. It is not known what factors predispose some tumours to be
aggressive and others not, but various growth factors and hormones have been
implicated. These include transforming growth factor α and β (TGFα/β), epidermal
growth factor (EGF), fibroblast growth factor 8 (FGF-8), insulin-like growth factors
(IGFs), Vitamin D and Thyroid hormone (Russell et al., 1998) (see Table 1.1). These
13
Chapter 1
Table 1.1: Role of growth factors in prostate cancer and metastasis
(adapted from Russell et al., 1998)
Regulatory
Factors
Expression and Regulation in
Prostate Cancer
TGFβ
Up-regulated in cancer; expression
correlates with responsiveness to
androgens; associated with abnormal
growth; autocrine regulation;
sensitivity to inhibition lost with
tumour progression
Stimulates LNCaP tumour formation;
expression increased in androgenindependent cells; autocrine regulation
by cancer cells
Over-expressed in cancer; promoter of
tumour growth; increased levels
correlate with disease progression;
loss of androgen regulation in
androgen-independent cell lines
Autocrine regulation; dysregulation of
IGFBP production
FGF-8
VEGF
IGFs/IGFBPs
EGF/TGFα
BMP
Up-regulated in cancer; autocrine
regulation
Present in varying amounts in cell
lines
Possible Role in
Metastasis/Cancer
Progression
Causes osteoblast migration,
angiogenesis,
immunosuppression
Regulates protease expression
(uPA, collagenase); highly
angiogenic
Paracrine mediator of tumour
angiogenesis; potential
promoter of metastasis
May promote bony metastasis;
PSA cleaves IGFBP-3/IGF-1
complex releasing bioactive
IGF-1
Induces tumour proteases
(uPA); can regulate IGF axis
May be produced at metastatic
sites; stimulates bone
formation
TGFβ: Transforming growth factor β; FGF: Fibroblast growth factor 8; VEGF: Vascular endothelial growth
factor; KGF: Keratinocyte growth factor; IGF: Insulin-like growth factor; IGFBP: Insulin-like growth factor binding
protein; EGF: Epidermal growth factor; TGFα: Transforming growth factor α; IL-6: Interleukin 6BMP: Bone
morphogenetic protein; uPA: urokinase plasminogen activator
14
Chapter 1
growth factors have also been shown to regulate a number of genes highly expressed in
the prostate, some of which include members of the human kallikrein gene family.
1.3.2 Estrogens
Both estrogens and androgens are individually capable of altering the normal growth of
the prostate, however, separately they do not induce prostatic malignancy. It has been
shown that, in combination, androgens and estrogens can lead to dysplasia, premalignant
and malignant changes to the cells of the prostate (Ho et al. 1995, Wang & Wong 1998,
Wang et al. 2000, Hayward et al. 2001). As neither hormone by itself is capable of
inducing malignant changes in the prostate, the balance between the hormones is critical,
in both normal function and in disease.
1.3.3 Vitamin D
Evidence suggests that dietary factors can affect the incidence of prostate cancer. A
deficiency in 1,25-dihydroxyvitamin D (vitamin D3) has been proposed to increase the
risk of prostate cancer (Schwartz and Hulka, 1990). In LNCaP cells (an established
androgen-dependent prostate cancer cell line), exposure to vitamin D3 can neutralize the
proliferative effects of androgens, suggesting that it is a strong inhibitor of epithelial cell
proliferation (Esquenet et al., 1996, Leman et al., 2003). It has been found that in
LNCaP, DU-145 and PC-3 cells (both DU-145 and PC-3 cells are established androgenindependent prostate cancer cell lines), vitamin D3 can up-regulate expression of IGFBP-6 mRNA in a dose dependent manner (Drivdahl et al., 1995), indicating that it may
modulate growth via the IGF axis (Leman et al., 2003). Furthermore, proliferation of
prostate epithelial cells by vitamin D3 is accompanied by an increase in insulin-like
growth factor binding protein-3 (Sprenger et al., 2001). Danielpour and co-workers
(1994) found that the inhibitory effects of vitamin D3 may actually be lost in late-stage
prostate cancer. These results suggest that vitamin D3 may be involved in prostate
cancer at both the early androgen dependent phase and the late androgen independent
phase.
15
Chapter 1
1.3.4 Thyroid Hormone
Triiodothyronine (T3) has been shown to induce a proliferative response by LNCaP cells
(Esquenet et al., 1995). Furthermore, T3 has been defined as one of the most critical
components to support growth of LNCaP cells in serum-free defined medium (Hedlund
and Miller, 1994). Thyroid hormone acts via a nuclear T3 receptor to cause regulation of
specific genes in much the same way as androgens modulate their effects on gene
transcription.
Recent studies have demonstrated the interactive effects of
triiodothyronine (T3) and androgens on prostate cell growth and gene expression (Zhang
et al., 1999). Zhang and co-workers (1999) found that triiodothyronine, in the absence
of androgens, repressed the expression of KLK2. Androgens, T3 or a combination of the
two produced a dose dependent up regulation of PSA. It was also found that T3 alone
showed pronounced growth enhancement in a dose-dependent manner. Yet, in the
presence of androgens, higher T3 concentrations were required to produce additional
proliferative effects.
1.3.5 TGFβ
In the nondiseased prostate, TGFβ inhibits proliferation and induces apoptosis in
prostatic epithelia, thus providing a mechanism to maintain epithelial homeostasis in the
prostate (Danielpour, 1999; Lee et al., 1999). As prostatic epithelial cells undergo
malignant transformation, two major events occur regarding TGFβ action: the loss of
expression of functional TGFβ receptors, and the overproduction of TGFβ in malignant
cells as prostate cancer progresses (Kim et al., 1996; Sintich et al., 1999). This results
in a growth advantage to malignant cells over their benign counterparts due to the loss of
the inhibitory effect of TGFβ, via the lack of functional receptors. The overproduction
of this growth factor has a multitude of adverse consequences. In the bone environment,
TGFβ exerts mitogenic effects on osteoblasts, which express a plethora of growth
factors (Watts & Ware, 1992). Furthermore, TGFβ has the capacity to modulate MMP
production (Sehgal et al., 1996), to stimulate adhesion of prostate cancer cells to bone
cells (Kostenuik et al., 1997) and has been linked to the process of epithelialmesenchymal transition (Hay, 1995), thereby providing a possible role for TGFβ in the
metastatic process. Recently, studies have suggested that TGFβ-mediated apoptosis can
16
Chapter 1
actually be enhanced by androgens through specific mechanisms involving cell cycle
and apoptosis regulators and provides initial evidence on the ability of physiological
levels of androgens to stimulate the intrinsic apoptotic potential of prostate cancer cells.
It was concluded that the study provided evidence for the priming of prostate cancer
cells for maximal apoptosis induction, during hormone-ablation therapy (Bruckheimer &
Kyprianou, 2001).
1.3.6 EGF and TGFα
EGF and TGFα are two structurally and functionally related peptide growth factors
found in prostatic fluids. Both EGF and TGFα expression are up-regulated in human
cancers. Increased expression of EGF/TGFα and the EGF receptor has been linked to
prostate cancer development as evidenced by raised protein levels of both factors in
prostate cancers in comparison with benign tissue (Harper et al., 1993; Glynne-Jones et
al., 1996; Olapade-Olaopa et al., 2000). Furthermore, their expression has been
associated with prostate cancer cells undergoing androgen independent progression
(Schuurmans et al., 1989; Chung et al., 1992). Studies utilising PC-3 cells in a Boyden
chamber microinvasion assay indicate that EGF enhances prostate tumour cell invasion
(Jarrad et al., 1994). Furthermore, Torring and colleagues (2000) have found that a
selective up-regulation of a subclass of ligands of the EGF-system in androgenindependent prostate cancer cell lines suggests this could be a mechanism to escape
androgen dependence in prostate cancer.
1.3.7 IGFS
There are two insulin-like growth factor peptides, IGF-I and IGF-II, two cell surface
receptors and at least six specific high affinity binding proteins, IGF-BP-1 through IGFBP-6, that regulate IGF availability and are in turn regulated by a group of IGF-BP
proteases that cleave IGF-BPs to modulate IGF action. In the nondiseased prostate,
IGFs are produced only by stromal cells. Studies have shown that the LNCaP cell line
proliferates in response to IGF-I but does not produce it. However, this proliferative
effect occurs only in synergy with dihydrotestosterone (DHT) (Iwamura et al., 1993).
The IGF axis in prostate cancer is complex and IGFs appear to have an important role in
17
Chapter 1
the development of prostate cancer. PSA, which is up-regulated by androgens, can
cleave IGF-BP-3 (Sutkowski et al., 1999) which could release IGFs locally to stimulate
prostate cancer cell growth. It has also been demonstrated that PSA can cleave IGFBP-4
in addition to IGFBP-3, but not IGFBP-2 and –5, whereas hK2 cleaved all of the
IGFBPs much more effectively, and at concentrations far lower than those reported for
other IGFBP-degrading proteases (Rehault et al., 2001). A potential role for the IGFs in
prostate cancer progression is in the development of bone metastases. Both IGF-I and
IGF-II mRNA transcripts have been detected in non-diseased human osteoblast-like
cells (Rajah et al., 1996) and appear to have an important role in bone formation
(Chevally et al., 1996). Interestingly, studies have shown that factors that decrease the
activity of IGF-BP-3, such as dexamethasone, also inhibit bone formation (Chevally et
al., 1996), indicating an important potential role for IGF-BP-3 in the formation of bone
metastasis in advanced prostate cancer.
1.3.8 FGF
The fibroblast growth factors (FGFs) are a family of nine peptides which are expressed
in the prostate at varying levels (Benharroch and Birnbaum, 1990, Rosini et al., 2002,
Gnanapragasam et al., 2003, Huss et al., 2003). The FGFs are mitogens and regulate
extracellular matrix production and contribute to angiogenesis in prostate tumours
(Polnaszek et al., 2003). In humans FGF-8 is involved in the pathogenesis of prostate
cancer, while the role of FGF-2 in prostate cancer development has only been observed
in rats (Mansson et al., 1989). FGF-8 is abundant in the prostate and is thought to be
produced in an autocrine manner by nondiseased stromal cells of the prostate. As
prostate cancer progresses, the production of FGF-8 becomes androgen independent and
is regulated in an autocrine fashion by prostate cancer epithelial cells (Geller et al.,
1994). FGF-8 regulates ECM production and by acting on endothelial cells to promote
tumour angiogenesis, its production and secretion enhances the metastatic capacity of
the tumour (Greene et al., 1997, Polnaszek et al., 2003). Furthermore, the contribution
of FGF-8 to tumour progression has been shown in patients with elevated levels of FGF8 and who are suffering from advanced prostatic disease (Cronauer et al., 1997).
18
Chapter 1
1.3.9 VEGF
Vascular endothelial growth factor (VEGF) is a cytokine that plays an important role in
tumour angiogenesis. VEGF is over-expressed in many human cancers, and patients
with metastatic prostate cancer have higher plasma VEGF levels than patients with
localized disease or healthy controls (Duque et al., 1999). VEGF has been shown to act
upon two tyrosine kinase family receptors: c-fms-like tyrosine kinase (Flt-1) and foetal
liver kinase. Widespread distribution of VEGF receptor Flt-1 in BPH, PIN and prostate
cancer specimens suggests that VEGF function in prostate is not restricted to endothelial
cells and angiogenesis. However, since the receptor is lost in prostate cancer cells and
with tumor dedifferentiation, these yet unknown effects of VEGF on epithelial cells are
obviously suppressed with malignant transformation (Hahn et al., 2000). A recent study
(West et al., 2001) has found a correlation of VEGF expression with fibroblast growth
factor-8 expression and clinical parameters in human prostate cancer. In particular,
VEGF immunoreactivity in both malignant epithelium and adjacent stroma is
significantly associated with high tumour stage. VEGF expression also correlated with
increasing serum PSA levels and is significantly associated with Gleason score.
Furthermore, cases showing positive VEGF immunoreactivity in the stroma had
significantly reduced survival rate compared to those with negative staining.
Cases
with tumours expressing both FGF-8 in the malignant epithelium and VEGF in the
adjacent stroma had a significantly worse survival rate than those with tumours negative
for both, or only expressing one of the two growth factors (West et al., 2001).
Interestingly, PSA has recently been shown to have antiangiogenic activity by inhibiting
endothelial cell proliferation, migration and invasion. Additionally, PSA inhibited
endothelial cell responses to FGF-2 and VEGF (Fortier et al., 1999).
1.4 PROSTATE CANCER CELL MODELS
To study prostate cancer in vitro, a number of approaches have been developed, which
include organ explant cultures, primary cell lines and established cell lines. The
established cell lines, LNCaP, PC-3 and DU145 are the most widely used prostate
cancer cell lines and multiple studies have been undertaken to characterise these cell
lines. These cell lines are epithelial in origin and were cloned from human metastatic
19
Chapter 1
prostate tissue. Other prostate cancer cell lines which have more recently been
developed, and were used in this study, include the RWPE1 and RWPE2 cell lines and
the LNCaP C4 series sublines.
The RWPE1 cell line is a non-invasive, non-malignant prostate epithelial cell line which
was established from non-neoplastic adult human prostatic epithelial cells immortalised
with human papillomavirus 18 (Bello et al., 1997). Cells from the RWPE1 cell line
were further transformed by v-Ki-ras to establish the RWPE2 cell line. The RWPE2
cells form tumours in nude mice and are also invasive using an in vitro invasion assay,
whereas the RWPE1 cells do not (Bello et al., 1997). The RWPE1 cells, which show
many normal cell characteristics, and the malignant RWPE2 cells, provide useful cell
culture models for studies on prostate disease.
The most commonly utilised cell line is LNCaP, and until recently, it was the only cell
line that was both androgen-sensitive and expressed PSA (Horoszewicz et al., 1983). It
contains a responsive androgen receptor, and although a mutation at codon 868
(Threonine to Alanine) affects its steroid-binding specificity for other steroids, this
change only slightly affects androgen action (Veldscholte et al., 1990). Unfortunately,
when the parental LNCaP line is injected into nude mice, it rarely produces metastases.
The C4 series of LNCaP sublines were derived from the LNCaP cell line by co-culture
with the human bone fibroblast cell line (MS) in male athymic mice (Chung et al.,
1997). As the C4 series of cell lines progress, they become increasingly metastatic and
androgen insensitive. The C4 series consists of the C4 (primary tumour), C4-2 (lymph
node metastasis) and C4-2B (bone metastasis derivative of the C4-2 cell line).
The DU145 cell line was isolated from a human brain metastasis (Stone et al., 1978) and
the PC-3 cell line was established from a bone metastasis (Kaighn et al., 1979). They
are both androgen resistant lines and have metastatic potential when inoculated in vivo
(Wu et al., 1998). Neither expresses PSA or the AR, which is still common to many
20
Chapter 1
androgen-independent prostate cancers.
However, these cell lines may reflect the
characteristics of the most advanced form of prostatic disease (Navone et al., 1998).
In summary, the cell lines discussed above, which were used in this study, provide a
good theoretical model to characterise the expression and regulation of genes which may
be important to the progression of prostate cancer.
1.5 PROTEASES ASSOCIATED WITH PROSTATE CANCER
Proteolytic enzymes, along with cell adhesion molecules, growth factors, matrix
molecules and cytokines, work together in complex pathways to determine the
metastatic potential of tumour cells. The molecular process of metastasis has been
described as a series of events progressing from initial invasion involving degradation of
ECM components, adhesion to and degradation of the underlying basement membrane,
angiogenesis, intravasation into blood or lymph vessels, followed by tumour cell
extravasation and proliferation in a specific secondary organ (Figure 1.3) (reviewed in
Staff, 2001). As ECM degradation is the first step in the metastatic cascade, dissolution
of ECM components is a vital function of malignant cells which is enabled by
expression and secretion of matrix-degrading proteases (Librach et al., 1991;
Behrendtsen et al., 1992; Edwards and Murphy, 1998).
Two protease systems
responsible for the proteolysis of the ECM, matrix metalloproteases (MMPs) and
uPA/plasminogen system will be discussed briefly, while the kallikrein family of serine
proteases will be reviewed in greater detail.
1.5.1 Matrix Metalloproteases
The MMPs are a family of proteolytic enzymes with each member specialising in the
degradation of various constituents of the stroma and basement membrane (Liotta and
Stetler-Stevenson, 1991). All MMPs are synthesised as zymogens (pro-MMPs) and
require extracellular activation. MMPs and their endogenous inhibitors, the tissue
inhibitors of metalloproteases (TIMPs), are known to play a crucial role in tumour
invasion and metastasis, presumably by destroying the integrity of the basement
membrane enabling cancer cells to invade normal tissue and facilitate secondary tumour
21
Chapter 1
Figure 1.3 Cellular Events Leading to Metastasis
Reproduced and modified from Zetter, 1998.
22
Chapter 1
deposits. The balance between MMP activity and the availability of TIMPs is a key
factor associated with tumour progression. In the malignant prostate, an imbalance
exists between MMPs and their natural inhibitors (Brehmer et al., 2003, Daja et al.,
2003, Lichtinghagen et al., 2003, Sehgal et al., 2003) with high levels of MMPs and low
levels of TIMPs expressed by prostate cancer cells. Additionally, prostate cancer cell
lines that express high levels of MMPs frequently metastasise to the bone and the lungs
(Lokeshwar, 1999).
1.5.2 Urokinase-type Plasminogen Activator
Proteolytic activity essential for stromal invasion by tumour cells can also be mediated
by the plasminogen activation system via urokinase plasminogen activator (u-PA)
induced proteolysis of the extracellular matrix. Plasmin is an important serine protease
bioactive in almost every physiological and developmental system (Festuccia et al.,
1998). Its expression has implications in cancer as it is able to digest ECM molecules
and activate latent metalloproteases and growth factors (Frenette et al., 1997b). Plasmin
is formed by the hydrolysis of plasminogen by plasminogen activators, one of which is
urokinase-type plasminogen activator (uPA; Lijnen et al., 1986). Components of this
proteolytic cascade are found in many invasive tumours including hormone dependent
malignancies such as breast, cervical, endometrial and prostate cancer (Achbarou et al.,
1994; Festuccia et al., 1995; Rabbani et al., 1995; Ferno et al., 1996; Fisher et al., 2000;
Riethdorf et al., 1999; Tecimer et al., 2000; Tecimer et al., 2001). Furthermore, overexpression of uPA has also been highly correlated to prostate cancer progression
(Achbarou et al., 1994; Festuccia et al., 1995; Rabbani et al., 1995).
1.5.3 The Human Kallikrein Family
Another family of enzymes that are implicated in the initiation, progression and
metastasis of solid tumour cancers, are the kallikreins.
Human tissue kallikreins
1
(KLKs ) are members of a multigene family of serine proteases that cleave specific
polypeptide precursors to release their bioactive forms (Clements, 1989; Riegman et al.,
1992). A major structural characteristic of all serine proteases is the non-contiguous
1
The standard nomenclature for the Kallikreins is KLK1-15 for the genes and hK1-15 for the human
proteins or enzymes (Diamandis et al., 2000a).
23
Chapter 1
catalytic triad, His – Asp – Ser, which is essential for catalytic activity. This triad is
fully conserved in the kallikrein gene family. Until recently the human gene family
contained only three members: KLK1, KLK2 and KLK3 which encode the proteins,
tissue kallikrein (hK1), glandular kallikrein (hK2) and prostate-specific antigen
(PSA/hK3) (Fukushima et al., 1985; Rittenhouse et al., 1998). Several groups, noting
that the kallikrein gene family in other species was notably larger than that of humans
(mouse with 26 genes and the rat, 13), undertook studies to estimate the size of the
human family.
Using Southern analysis, several groups predicted the family would contain four
members (Baker and Shine, 1985; Fukushima et al, 1985; Schedlich et al, 1987;
Reigman et al, 1989). However, Murray and co-workers (1990) using a monkey KLK
cDNA probe on human genomic Southerns detected nineteen potential members. Not
surprisingly, much research has been conducted in order to determine the true size of the
human kallikrein gene family. Our group and others have contributed to the expansion
of this family (Nelson et al., 1999; Stephenson et al., 1999; Yousef et al., 1999a;
Yousef et al., 1999b; Harvey et al., 2000; Hooper et al., 2001; Yousef et al., 2001b).
The kallikrein gene family in humans now includes 15 genes (KLK1-15) in a 300kb
region located on chromosome 19q13.4 (Figure 1.4) (reviewed in Clements et al., 2001).
The twelve new members, along with the classical 3 kallikreins, share significant
similarities.
Firstly, they display notable sequence identity (30-80%) at the DNA and
protein level (Harvey et al., 2000; Yousef et al., 2000c). Furthermore, the structural
organisation of the KLK genes is conserved, consisting of five coding exons of similar or
identical size (although some members contain one or more 5’ untranslated exons) and
fully conserved intron phases (Riegman et al., 1992; Stephenson et al., 1999; Harvey et
al., 2000; Yousef et al., 2000c). Interestingly, in humans many of the kallikrein genes
have multiple splice variants.
All genes encode for putative secreted pro-serine
proteases containing the conserved catalytic triad of amino acid residues. Removal of
the pro-region is required for activation and many appear to be regulated by steroid
hormones (reviewed in Diamandis et al., 2000a; Yousef et al., 2001a).
24
Chapter 1
Centromere
Telomere
19q13.3 13.4
KLK3
KLK1
KLK15
KLK8
KLK12
KLK9
KLK6
KLK13
KLK4
KLK10
KLK5
KLK14
KLK2
KLK7
KLK11
Figure 1.4 The human kallikrein locus
The position of the 15 kallikrein encoding genes on the KLK locus are marked. KLK1
and KLK4 to KLK15 are transcribed telomere to centromere, whereas KLK2 and KLK3
are transcribed in the opposite direction. Reproduced and modified from (Clements et
al., 2001).
25
Chapter 1
As most of these genes have only recently been identified, their characterisation with
respect to cancer involvement is currently under investigation. Several studies have
provided some insight into potential roles of the newer family members in the
pathogenesis and/or progression of cancers and will be briefly outlined here. KLK6
(Protease M/Zyme/Neurosin/PRSS9) was first discovered due to its dramatic down
regulation in metastatic breast carcinoma cells, in comparison to primary cancer cells or
normal breast epithelial cells (Anisowicz et al, 1996).
Additionally, KLK6 is
upregulated by steroid hormones in the breast cancer cell line BT-474 (Yousef et al.,
1999b) and is also highly expressed in ovarian cancer (Anisowicz et al., 1996; Yousef et
al., 1999b). K6, the encoded protein, can hydrolyse amyloid precursor protein and
therefore is suggested to be important in the deposition of amyloid plaques in
Alzheimer’s disease (Little et al, 1997). KLK13 (previously known as KLK-like 4), is
expressed in breast, and preliminary data demonstrate that KLK13 is down-regulated in
breast cancer tissue and cell lines at the mRNA level and is up-regulated by androgens,
progestins and to a lower extent by estrogens (Yousef et al., 2000b). KLK10 (NES-1)
expression is also down-regulated in breast and prostate cancer tissues, and is suggested
to be a novel tumour suppressor gene (Liu et al., 1996; Goyal et al., 1998; Luo et al.,
2000). KLK7 and KLK8 have been reported to be overexpressed in ovarian carcinomas
(Underwood et al., 1999). The isolation of other kallikreins from specific tissues such as
the skin (KLK5, KLK7, KLK11) and brain (KLK6, KLK8, KLK11) suggests a potential
role at these sites (reviewed in Diamandis et al., 2000a; Clements et al., 2001). In
addition to these sites, KLK6-KLK13 have been shown to be highly expressed in the
pancreas (Harvey et al, 2000) also suggesting an important role in pancreatic function
for these enzymes. As mentioned above, the expression of several KLK genes in breast,
ovarian and prostate cancer tissues or cell lines demonstrates that many members of this
family are associated with hormone-dependent cancer (Diamandis et al., 2000a). Those
members of the family that are highly expressed in the prostate include KLK2, KLK3 and
KLK4 and will be reviewed in greater detail below.
26
Chapter 1
1.5.3.1 KLK2 and KLK3
Both KLK2 and KLK3 are highly expressed in the prostate, and were originally thought
to be prostate specific, however they have since been detected in other tissues (Lundwall
& Lilja, 1987; Hsieh et al., 1997; Riegman et al., 1988; Rittenhouse et al., 1998;
Magklara et al., 2000). The glandular epithelial cells of the prostate produce both K2
and PSA. PSA is secreted into the ejaculate where it plays an important role in semen
liquefaction by digesting the seminal proteins, semenogelin and fibronectin, allowing
mobility of sperm after ejaculation (Lilja, 1985). Disruption of the prostatic architecture,
as a result of abnormal cellular growth, releases PSA into the circulation at high
concentrations, thereby providing the basis for the PSA diagnostic test for prostate
cancer (Stamey et al., 1987; Oesterling, 1991). PSA has undergone scrutiny as to
whether it is the best possible marker. There is much evidence showing that serum PSA
is elevated in BPH as well as prostate cancer (Daher & Beaini, 1998). Additionally
serum PSA levels can be low in cancer suggesting that there is a need for a more
discriminating marker that is more sensitive and specific for malignant disease. To
address this, modifications of the PSA test have improved the cancer specificity by
utilising parameters such as free/total PSA ratio (Veltri and Miller, 1999), PSA density
(Benson et al., 1992) and PSA velocity (Carter et al., 1992; Carter, 1997; Carter &
Pearson, 1997). In the ‘grey zone’ of 4-10ng/ml PSA, the ratio of free to total PSA has
been shown to improve the discrimination between prostate cancer and BPH and higher
levels of free PSA correlate to a lower risk of prostate cancer (Catalona et al., 1998;
Makinen et al., 2001, Tornblorn et al., 2001). Recent studies by Mikolajczyk and
colleagues (2001) have provided evidence for the use of a truncated form of precursor
PSA (pPSA) as a more specific serum marker of prostate cancer. The truncated
precursor PSA consists of PSA with a serine-arginine pro-leader peptide, ([-2]pPSA),
instead of the normally expressed 7 amino acid pro-leader peptide. In vitro activation
studies showed that human kallikrein 2 and trypsin readily activated full-length pPSA
but were unable to activate [-2]pPSA to mature PSA. Thus, [-2]pPSA, once formed, is a
stable but inactive isoform of PSA (Mikolajczyk et al., 2001). Recent evidence has
shown that K2 is more associated with prostate tumours than PSA and is highly
expressed in poorly differentiated cancer cells (Darson et al., 1997, Charlesworth et al.,
27
Chapter 1
1997; Lilja, 2001).
Furthermore, it has been suggested that K2 may be a potential
serum marker for predicting the organ confined versus non-organ confined growth of
prostate cancer (Haese et al., 2000; Recker et al., 2000; Becker et al., 2000). This
suggests that there is a potential role for K2 as a more specific diagnostic/prognostic
marker of late stage prostate cancer than PSA.
PSA been suggested to mediate invasion and metastasis via proteolytic cleavage of the
basement membrane proteins, fibronectin and laminin (Webber et al., 1995). Webber
and co-workers (1995) demonstrated this proteolytic activity of PSA using an invasion
assay and a reconstructed basement membrane (Matrigel). Despite these findings, the
precise function of PSA in prostate cancer biology is unknown; therefore several studies
are examining its role in prostate cancer. The current literature either reports that PSA is
a beneficial molecule with tumour suppressor activity or that PSA has deleterious effects
in prostate, breast and possibly other cancers. PSA is a favourable prognostic marker in
breast cancer and its production is generally reduced in breast cancer compared with
normal or hyperplastic breast tissue. Furthermore, women with PSA-positive tumours
live longer and relapse less frequently (Yu et al., 1995; Yu et al., 1998). Prostate
cancer cell lines that have been transfected with KLK3 cDNA have been shown to
become apoptotic, have decreased proliferation rates and give rise to tumours with
decreased metastatic potential (Balbay et al., 1999). In addition to this, PSA appears to
have potent anti-angiogenic activity, and hence inhibits tumour formation (Fortier et al.,
1999). In summary, these findings suggest that PSA might act as a tumour suppressor or
an inducer of apoptosis.
On the other hand, evidence suggests that PSA may be
deleterious in cancer due to its ability to cleave the IGF binding protein, IGFBP-3, thus
liberating IGF-1, which is a mitogen to prostatic stromal and epithelial cells (Cohen et
al.,1992; Sutkowski et al., 1999). Additionally, PSA may activate latent TGF- β,
stimulate cell detachment and therefore facilitate tumour spread (Killian et al., 1993).
Studies focusing on hK2 have shown that it may initiate a proteolytic cascade by
activating urokinase plasminogen activator (uPA), which itself is highly associated with
prostate cancer progression. uPA converts plasminogen to plasmin which then acts
directly to degrade the extracellular matrix (Frenette et al., 1997).
28
Kumar and
Chapter 1
colleagues (1997) have detected an additional function of hK2, which is the cleavage of
pro-PSA to generate the enzymatically active PSA protein. The interactions of these
enzymes occur in vitro and may be important in proliferation and invasion of cancer
cells by the activation of growth factors or extracellular proteases, or degradation of the
extracellular matrix (Clements, 1998). As hK2 has an important in vivo regulatory effect
on PSA activity and PSA is known to facilitate prostate cancer cell invasion (Webber et
al., 1995), hK2 therefore indirectly aids in this process also.
In the prostate, both KLK2 and KLK3 are regulated by androgens both at the level of
gene expression and enzyme activity. The androgen response elements (AREs), which
have been reported in the promoter of the human KLK2 and KLK3 genes, are necessary
for transcriptional regulation by androgens (Riegman et al., 1991; Murtha et al., 1993;
Young et al., 1995). Thyroid hormone and the growth factors (EGF, TGF) also affect
KLK2 expression in the human prostate tumour LNCaP cell line (Henttu and Vihko,
1993; Esquenet et al., 1995; Shan et al., 1997). Recently, Zhang and co-workers (1999)
demonstrated the interactive effects of triiodothyronine (T3) and androgens on prostate
cell growth and gene expression. It was shown that T3 alone showed pronounced
growth enhancement in a dose-dependent manner. Yet, in the presence of androgens,
higher T3 concentrations were required to produce additional proliferative effects. They
also demonstrated that T3, in the absence of androgens, suppressed the expression of
KLK2 and androgens, T3 or a combination of the two produced a dose dependent up
regulation of the PSA protein. There is also evidence which suggests that the PSA gene
is up-regulated by T3 at the transcriptional level via a functional T3-responsive element
(TRE) in the 5’ promoter region of the gene (Zhu & Young, 2001).
1.5.3.2 Human KLK4
1.5.3.2.1 Expression
Recently, a new member of the human kallikrein gene family, KLK4, was discovered
and characterised by several groups (Nelson et al., 1999, Stephenson et al., 1999,
Yousef et al., 1999, Hu et al., 2000b, Korkmaz et al., 2001). Like KLK2 and KLK3,
KLK4 mRNA is most abundantly expressed in the epithelial cell of the normal and
29
Chapter 1
cancerous prostate. Also known as prostase, serine protease 17 (PRSS17), kallikrein like
protease 1 (KLK-L1), androgen-regulated message 1 (ARM1) and enamel matrix serine
protease 1 (EMSP1), the KLK4 gene sequence consists of five coding exons, and is
predicted to encode a 254 amino acid protein that includes the conserved amino acid
residues (His41–Asp90–Ser180) required for serine protease function.
It also has an
amino-terminal pre-propeptide sequence, indicating a potential secretory function
(Nelson et al., 1999). Consistent with the majority of the human kallikreins, KLK4 has
potential trypsin-like substrate specificity; a function denoted by an aspartate six
residues before the catalytic serine.
Expression studies have demonstrated KLK4 expression in a wide variety of human
tissues. These include prostate, endometrium, and breast cancer tissues, testis, adrenal,
kidney, uterus, thyroid and mammary tissue (Nelson et al., 1999, Stephenson et al.,
1999, Yousef et al., 1999,). KLK4 expression has been identified in the endometrial
cancer cell lines, HECA, HEC1B, Ishikawa, KLE and RL95-2; the ovarian cancer cell
lines, OVCAR-3 and OAW42; the breast cancer cell line, BT-474; and the LNCaP
prostate cancer cell line (Nelson et al., 1999, Stephenson et al., 1999, Yousef et al.,
1999, Obiezu et al., 2000, Dong et al., 2001, Myers and Clements, 2001, Korkmaz et al.,
2001). KLK4 has recently emerged as a potential marker for ovarian cancer (Dong et
al., 2001). It was demonstrated that KLK4 is highly expressed in late stage serous
epithelial-derived ovarian carcinomas compared to normal ovaries, mucinous epithelial
tumours and granulosa cell tumours (Dong et al., 2001). It has also been established that
KLK4 expression acts as an independent unfavourable prognostic factor in patients with
grade 1 and 2 ovarian tumours (Obiezu et al., 2001). This study found that patients with
ovarian tumours positive for KLK4 expression had an increased risk for relapse and
death. Clearly, KLK4 expression is associated with hormone dependent cancers and
may be a useful biomarker in ovarian cancer.
As KLK4 is most abundantly expressed in the prostate (Nelson et al., 1999), a number of
studies have been undertaken to more fully examine its expression pattern in prostate
tissue sections and in prostate cancer cell lines. A study of KLK4 expression in prostate
30
Chapter 1
tissues indicated that KLK4 mRNA levels were similar in all normal, benign and tumour
samples examined. However, only 3 normal and 3 BPH samples were compared to 24
prostate tumour samples (Day et al, 2002). Additionally, in a study by Obiezu and
colleagues (2002) it was found that hK4 concentrations were highest in healthy prostate
extracts compared with cancerous extracts, using a sandwich-type immunoassay (Obiezu
et al., 2002). The immunoassay developed by this group detected hK4 in 10 out of 21
matched normal/cancer samples. The authors concluded that although KLK4 mRNA is
readily detectable by RT-PCR, hK4 protein is present in some prostatic tissue extracts
but at relatively low concentrations and that the protein is either not synthesised
efficiently
or
is
degraded
quickly.
In
contrast
to
these
two
studies,
immunohistochemical analysis of tissue sections within this laboratory have
demonstrated that hK4 is expressed to a greater degree in prostate cancer in comparison
to BPH sections (Ms L Bui, personal communication).
Due to the considerable
discrepancies between the results of this laboratory and others, it is clear that further
experimentation is required to more accurately quantitate KLK4/hK4 in both benign and
malignant prostate tissues.
1.5.3.2.2 KLK4 mRNA Variant Transcripts
Variant mRNA transcripts are a common feature of the human KLK family and have
been demonstrated for other members including KLK1 (Rae et al., 1999), KLK2
(Rittenhouse et al., 1998), KLK3 (Heuze et al., 1999, Tanaka et al., 2000, HeuzeVourc'h et al., 2003), KLK8 (Magklara et al., 2001), KLK11 (Mitsui et al., 2000) and
KLK13 (Yousef et al., 2000a). Thus far, four KLK4 variants in addition to the full length
KLK4 have been described (Obiezu and Diamandis, 2000, Dong et al., 2001, Korkmaz
et al., 2001, Myers and Clements, 2001) (Figure 1.5). It is important to note that the
KLK4 variants 2, 3 and 4 have premature stop codons that would lead to C-terminally
truncated hK4 proteins if translated (Figure 1.6). The proposed protein products of these
variants would not contain Ser207 of the catalytic triad and are therefore unlikely to
encode proteins with serine protease activity.
31
Chapter 1
ATG
Full Length
Exon 1
Exon 2
Exon 3
Exon 4
Exon 5
ATG
Exon 2
Exon 3
Exon 4
Exon 5
Variant 1
Exon 1 deletion
ATG
Exon 2
ATG
Exon 1
ATG
Exon 3
Exon 4
Exon 5
Variant 2
Exon 1 deletion
and Intron 3
insertion
ATG
Exon 2
Exon 3
Exon 4
Exon 5
Variant 3
Partial Intron 2
insertion
ATG
Variant 4
Exon 4 deletion
Exon 1
Exon 2
Exon 3
Exon 5
Figure 1.5 Schematic diagram of full length KLK4 and alternatively spliced mRNA
transcripts.
Full length KLK4 and four variant transcripts are illustrated. The sites of two potential
start codons are indicated by the ATG. Exons are represented as purple boxes, introns as
a line, and intron insertions are represented as green boxes. Dotted lines indicate that
Variants 3 and 4 may also exist as an exon 1 deleted form. The figure is not drawn to
scale.
32
Chapter 1
His
Met
Pre
Pro
Ser
Asp
Full Length
Mature
Met
His
Asp
Ser
Mature
Met His
Variant 1
Asp
Mature
Variant 2
His
Met
Pre
Variant 3
Pro
His
Met
Pre
Pro
Asp
Mature
Variant 4
Figure 1.6 Schematic diagram of full length and variant hK4 proteins.
Shown are the pre-pro-protein for full length hK4 and the truncated hK4 proteins
encoded from the KLK4 splice variants. The amino acids which comprise the catalytic
triad (His/Asp/Ser) are shown above the protein. The figure is not drawn to scale.
33
Chapter 1
The variant transcripts arise from alternative splicing with exon deletions and/or intronic
sequence inclusions. Figure 1.5 illustrates a schematic diagram of full length KLK4 and
its four alternative splice variants. Variant 1, which contains exon 2-5 is the most
dominant transcript in LNCaP and prostate cancer xenografts. This variant, which
contains only exons 2-5, is not secreted and has a distinct perinuclear cellular
localisation (Korkmaz et al., 2001). Conversely, full length KLK4 mRNA is only very
lowly expressed in the LNCaP prostate cancer cell line, but is also expressed in normal
and malignant prostate tissue (Korkmaz et al., 2001, Obiezu et al., 2000). In addition to
normal and malignant prostate tissues, prostate cancer xenografts and prostate cancer
cell lines, some of these KLK4 variant mRNAs are expressed in endometrial and ovarian
cancer cell lines (Obiezu and Diamandis, 2000, Dong et al., 2001, Korkmaz et al., 2001,
Myers and Clements, 2001).
Interestingly, as described above, results from our laboratory show an association of
KLK4 transcript expression with ovarian cancer progression (Dong et al., 2001). It
remains to be established whether any of these KLK4 variants may be differentially
expressed between benign and malignant prostate tissue and may therefore be of use in
providing a more discriminating marker for prostate disease.
1.5.3.2.3 Regulation
Since the report of KLK4 expression in the androgen dependent LNCaP cell line, a
number of studies assessing the regulation of KLK4 by androgens have been performed.
Nelson et al (1999) used the LNCaP line to evaluate KLK4 expression and found that 1
nM synthetic androgen, R1881, produced a 1.7 fold increase in expression in LNCaP
cells after 48 hours. A more recent study found that treatment of LNCaP cells with 10
nM R1881 for 24 hours resulted in an approximate 18 fold increase in KLK4 mRNA as
determined by Northern blot analysis (Korkmaz et al., 2001). In addition to the reported
up-regulation of KLK4 in the LNCaP model (Nelson et al., 1999, Korkmaz et al., 2001),
up-regulation of KLK4 has been demonstrated in the breast carcinoma cell line, BT-474,
by DHT (physiologically active androgen) as well as estradiol (Yousef et al, 1999).
Additionally, progestins were found to up-regulate KLK4 expression in BT-474 cells
34
Chapter 1
(Yousef et al., 1999). Expression of the exon 1 deleted KLK4 transcript has been
assessed in response to multiple hormones (Korkmaz et al, 2001). The exon 1 deleted
variant was found to be upregulated by androgen, estrogens, progestins and
dexamethasone but not by vitamin D3 or thyroid hormone in the LNCaP cell line after
24 hours of hormone stimulation (Korkmaz et al, 2001). As the prostate gland relies on
a variety of growth factors/hormones to maintain tumour growth, it is important to more
clearly define the effects of these growth factors/hormones on genes shown to be
important in prostate malignancies.
1.5.3.2.4 Functional Studies
hK4 exhibits most similarity to the pig enamel matrix serine protease, EMSP1 (78%
identity) (Nelson et al., 1999, Stephenson et al., 1999, Yousef et al., 1999), which
functions in the degradation of the enamel matrix as teeth develop (Fukae and Shimizu,
1974). EMSP1 is highly expressed in the early maturation stage of developing enamel
and has been suggested to degrade the organic matrix surrounding enamel crystallites,
which allows the enamel layer to fully mineralise (Scully et al., 1998; Hu et al., 2000a).
Interestingly, porcine KLK4 degrades recombinant amelogenin in vitro, supporting the
conclusion that KLK4 plays an important role in enamel protein degradation (Ryu et al.,
2002). Considering the protein similarity, it has been suggested that KLK4 is the human
homologue of this gene and may function in ECM degradation of the prostate
(Stephenson et al, 1999) and that hK4 may be involved in metastasis to bone, the
principal metastatic site of prostate carcinoma (Nelson et al, 1999). Considering KLK4
has a transcriptional response element for the bone specific transcription factor, Cbfa1,
upstream of its putative transcription start site (Hu et al., 2000a), a potential
physiological role of hK4 in the bone and/or tooth development could be expected.
Biochemical studies have proposed various enzymatic actions of hK4. For example, it
has been shown to completely degrade the seminal plasma protein, prostatic acid
phosphatase, but failed to cleave serum albumin, another protein from human seminal
plasma (Takayama et al., 2001). The authors suggested that hK4 may have a role in the
physiologic processing of seminal plasma proteins such as pro-PSA and PAP. PAP is a
35
Chapter 1
major component of prostatic fluid and has been shown to cleave human seminogelin I;
a key process required for the dissolution of the seminal clot allowing release of motile
sperm for fertilisation (Brillard-Bourdet et al., 2002). Therefore, it is possible hK4 may
play a role in the physiological processing of seminal plasma proteins during male
reproduction.
hK4 has also been implicated in the processes of migration and invasion due to its ability
to activate pro-PSA and single chain urokinase-type plasminogen activator (scuPA, prouPA) (Takayama et al., 2001). Prostate cancer cells are known to over-express uPA
(Achbarou et al., 1994, Van Veldhuizen et al., 1996,), which activates plasminogen to
generate plasmin (Lijnen et al., 1986), which in turn activates metalloproteases
(Stricklin et al., 1977, He et al., 1989) allowing them to digest ECM proteins, enabling
migratory cells to escape from the primary tumour site. Additionally, the digestion of
amelogenin in vitro by porcine K4 demonstrates its proteolytic cleavage of peptide
bonds (Ryu et al., 2002). This finding supports the notion that KLK4 may have a role in
tumour invasion and metastasis, where tumour cells must degrade extracellular matrix
molecules in order to gain access to the vasculature and subsequently form secondary
tumour deposits.
1.6 CONCLUSION AND RELEVANCE TO PROJECT
Prostate cancer is a disease that affects older males and is associated with significant
morbidity and mortality rates.
Unfortunately, current diagnostic strategies for the
detection of prostate cancer yield high numbers of false positive results, highlighting the
need for a more reliable, non-invasive screening method. Furthermore, current
therapeutic strategies carry considerable side effects or involve invasive surgery. The
treatment of prostate cancer with surgery and/or radiation therapy is often successful in
the early stages of disease; however, these treatments are associated with a high
morbidity. Treatment with androgen ablation therapy is often administered, although
difficulties arise when tumours escape from androgen regulation and become hormone
refractory. Prostate cancer is symptomless in the early stages and therefore many men
presenting with symptoms often have metastatic disease and a poor prognosis. As the
36
Chapter 1
cancer progresses and spreads to secondary sites, such as regional lymph nodes and
bone, treatment is essentially palliative. Therefore many studies are currently focusing
on biological molecules that may provide an insight into the pathogenesis and/or
progression of prostate disease in order to develop other therapeutic options or enhance
current diagnostic/prognostic approaches.
Two members of the kallikrein family of serine proteases, PSA and hK2, are important
biomarkers for prostate cancer diagnosis and increasing evidence suggests a functional
role for these proteins in progression of the disease. Although only a newly identified
member of the kallikrein family, KLK4 has emerged as an important gene in hormone
dependent cancers. It has recently been demonstrated that KLK4 transcript expression is
associated with ovarian cancer progression (Dong et al., 2001). Although there have
been limited studies examining the potential for KLK4 to act as a biomarker of prostate
disease, one study found no differences in KLK4 transcript expression between normal,
benign and malignant prostate tissue. However few tissue samples were used, therefore a
significant conclusion cannot be based on this finding. Considering two members of the
kallikrein family have already proven useful as diagnostic markers for prostate cancer
and that KLK4 is highly expressed in the prostate, it is logical to further examine the
potential of this gene as a new/adjunct biomarker of prostate disease.
Prostate cancer is a hormone dependent cancer as evidenced by its dependence upon
androgens in the initial phase. Although androgens are important in the early stage of
prostate to maintain the growth of the tumour, other growth factors are involved at this
stage also. Complex interactions occur between androgens, estrogens, IGFs, FGF-8 and
EGF until the disease progresses to an androgen insensitive phase, when these other
growth factors become increasingly important in sustaining the growth of the tumour.
Growth factors/hormones exert their effects on prostate growth and tumour progression
by regulating the expression of genes important in the tumourigenic processes, such as
KLK2 and KLK3.
Whilst a number of studies have assessed KLK4 regulation in
response to androgens in the prostate, it is important to confirm these studies and extend
them by examining other growth factors not previously assessed. In particular thyroid
37
Chapter 1
hormone and EGF are two such hormones/growth factors which have not been examined
with respect to KLK4 regulation.
There is accumulating evidence that KLK4/hK4 may play an important role in the
development and/or progression of prostate cancer. Although studies have implicated
hK4 in the processes of migration and invasion due to its ability to activate pro-PSA and
pro-uPA, and to degrade amelogenin, these suggestions have been made based on
biochemical assays. Thus, it is important to assess the role of hK4 in these malignant
processes in a cellular system.
Therefore, the overall aim of this project was to investigate the expression of KLK4
transcripts in prostate cancer and benign prostatic hyperplasia (BPH) to assess their
potential as cancer biomarkers; to further examine the hormonal/growth factor
regulation of KLK4/hK4 in prostate cancer; and to explore the functional consequences
of hK4 over-expression in prostate cancer progression using the PC-3 line as an in vitro
model system.
Thus, the specific aims of this project are:
1. To further characterise the expression of KLK4 mRNA transcripts in prostate cancer
and BPH tissues, in order to determine if differential expression exists in the two disease
states, using quantitative RT-PCR.
2.
To extend the known expression profile of KLK4 in prostate cancer cell lines
representing a spectrum of disease ranging from androgen sensitive through to androgen
insensitive and metastatic disease.
3. To assess the potential regulation of KLK4/hK4 in the androgen dependent prostate
cancer cell line, LNCaP, in response to androgens, thyroid hormone and epidermal
growth factor.
38
Chapter 1
4. To generate hK4 over-expressing PC-3 prostate cancer cell lines.
5. To determine the functional consequences of hK4 over-expression in the PC-3 cell
lines (from Aim 4), by investigating various cellular mechanisms of tumour progression,
which include:
(a) proliferation/growth rates
(b) chemo-invasion through a synthetic ECM (Matrigel)
(c) motility through the pores of a membrane barrier
(d) attachment to ECM molecules
(e) morphological changes.
39
CHAPTER TWO
MATERIALS AND METHODS
Chapter 2
2.0 INTRODUCTION
This chapter will outline the methods that have been used in one or more of the studies
reported in the Results section. All volumes are % w/v unless otherwise stated. General
chemicals and reagents of analytical grade were purchased from Ajax Chemicals
(Melbourne, Australia), BDH Merck (Kilsyth, Australia) or Sigma Chemical Company
(Castle Hill, Australia), unless stated otherwise.
2.1 MATERIALS AND METHODS
2.1.1
Cell Culture
The cell lines RWPE1, RWPE2, LNCaP, DU145, PC-3 and Saos-2 were obtained from
the American Type Tissue Culture Collection (Rockville, MD, USA). The LNCaP C4
series was obtained from Associate Professor Erik Thompson (St Vincent’s Institute of
Medical Research, Melbourne) and the Neonatal Foreskin Fibroblast (NFF) cells were
obtained from Dr Mark Hayes (Royal Children’s Hospital, Brisbane).
The general details of cell culture are summarized below; however, specific details
relating to particular experiments are outlined where applicable in the Results chapters.
2.1.1.1 Resuscitation of Cells from Liquid Nitrogen
Each ampoule of cryo-preserved cells was resuscitated from liquid nitrogen by rapidly
thawing at 37°C and transferring the cells to 40 ml of pre-warmed RPMI 1640 medium
(Invitrogen,
Brisbane,
Australia)
containing
penicillin/streptomycin/glutamine
(Invitrogen) and 10% Fetal Bovine Serum (FBS; CSL, Brisbane, Australia). Unless
otherwise stated, medium shall refer to RPMI 1640 medium, pre-warmed to 37°C,
containing 100 units/ml penicillin G sodium, 100 units/ml streptomycin sulphate, 0.3
mg/ml L-glutamine and 10% heat inactivated FBS. Cells were then pelleted by
centrifugation at 1000 g for 3 min at room temperature. The supernatant was aspirated
and the cells resuspended in 1 ml of medium and transferred to a T25 cm2 tissue culture
flask (Medos, Brisbane, QLD, Australia) containing 4 ml of medium. Cell lines were
cultured in an atmosphere of 95% air, 5% CO2 at 37°C in an IR Sensor Incubator
41
Chapter 2
(Quantum Scientific, Milton, QLD, Australia) until they were approximately 80%
confluent.
2.1.1.2 Routine Passaging of Cells
Cells were passaged at approximately 80% confluency. Spent medium was aspirated
from the cells and the cell monolayer was washed in pre-warmed sterile Phosphate
Buffered Saline (PBS) (Tissue culture grade; Oxoid, West Heidelberg, Vic, Australia)
followed by the addition of 1 ml of 0.05% trypsin/0.5 mM EDTA (Invitrogen). The
flask was then incubated at 37°C for approximately 5 min or until the cells detached
from the flask surface. An appropriate volume of medium (containing fetal calf serum
(FCS) to deactivate the trypsin) was then added to the flask. The cells were aliquoted at
the desired split (1:2 or 1:4) and the volume adjusted with medium and then incubated at
37°C.
2.1.1.3 Preparation of Cryo-Preserved Stocks
When stocks of preserved cell lines were used, new aliquots of cells were prepared to
replenish those resuscitated.
This involved passaging cells in the usual manner but
rather than aliquoting cells in medium after trypsin-treatment, they were resuspended in
10 ml of medium and transferred to a 15 ml tube. The cells were then pelleted by
centrifugation at 1000 rpm for 3 min. The supernatant was aspirated and the cell pellet
was resuspended in 1 ml of medium containing 10% dimethyl sulfoxide (DMSO;
Sigma, Castle Hill, NSW, Australia) and transferred to a cryovial. The cells were then
frozen slowly at -1°C/minute over several hours in an isopropanol-containing cryovessel
at -70°C. The frozen cells were then transferred to liquid nitrogen for long-term storage.
2.1.2
RNA Extraction
Total RNA from prostate tissues (prostate cancer and BPH) and prostate cancer cell lines
was extracted using the TRI Reagent (Sigma) according to the manufacturer’s
instructions. RNA from prostate tissue samples was extracted by Tara Veveris-Lowe.
Briefly, 1 ml of TRI Reagent was required per cell pellet from a T80cm2 culture flask
or per 60 mg of tissue. Cell/TRI Reagent preparations were homogenized by passing
42
Chapter 2
the mixture through a pipette and subsequent vortexing and tissue/TRI Reagent
preparations were homogenized using a Polytron (P-3000, Kinematica AG, Switzerland)
using several bursts of 18000-20000 rpm. Homogenates were incubated at room
temperature (RT) for 5 min to allow complete dissociation of nucleoprotein complexes,
followed by the addition of 0.2 ml of chloroform per ml of TRI Reagent to extract the
RNA. The mixture was shaken vigorously for 15 sec, left at room temperature for 3 min
and then centrifuged at 12000 x g for 15 min at 4°C. The aqueous phase was transferred
to a fresh tube and 0.5 ml of 100% isopropanol was added per ml of TRI Reagent to
precipitate the RNA. The mixture was incubated at -20°C for at least 2 hours before
being centrifuged at 12000 x g for 10 min at 4°C. The supernatant was discarded and
the precipitated RNA was washed by adding 1 ml of 75% ethanol per 1 ml of TRI
Reagent, vortexed and centrifuged at 7500 x g for 10 min at 4°C and the pellet left to
air dry at RT. The RNA pellet was dissolved in 20 µl of diethyl pyrocarbonate (DEPC)treated water.
The RNA was analysed for purity (A260/A280: ratio = 2.0) and
concentration (A260: O.D of 1 ≈ 40 µg/ml RNA) spectrophotometrically using a DU 640
Beckman spectrophotometer wavelength scan analysis from 220nm – 320nm. Samples
were then stored at -70°C until needed.
2.1.3
Polymerase Chain Reaction (PCR)
2.1.3.1 Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
DNAse treatment was carried out on RNA to be used for complementary DNA
(cDNA)/first strand synthesis to eliminate genomic DNA contamination. 10 units of
DNAse I (Roche) was incubated with 5 µg RNA for 1 hour at 37°C in a total volume of
10 µl. cDNA synthesis was carried out using 5 µg of DNAse I treated RNA. The RNA
was annealed to 100ng of oligo-dT primer by incubation for 10 min at 70°C and then
chilled on ice. First strand synthesis was achieved using 200 U Superscript II
(Invitrogen) in 10 mM DTT, 0.5 mM dNTPs, 5x first strand buffer (250 mM Tris-Cl pH
8.3, 375 mM KCl, 15 mM MgCl2) to a reaction volume of 20 µl for 90 min at 43°C.
The samples were then diluted 5-fold in sterile distilled water. All cDNA samples were
screened for genomic contamination by PCR amplification of β2-microglobulin, to yield
43
Chapter 2
a 249 bp PCR product. The primers used span a 615 bp intron, which would yield a
PCR product of 864 bp if genomic contamination was present.
PCR was performed using primers specific for the gene of interest. PCR reaction
mixtures contained 2 µl 10x PCR buffer (Roche; containing 15 mmol/l Mg2+), 0.25 U
Platinum Taq, 10 µM each of forward and reverse primer, 2 µl of 2 mmol/l dNTP mix
and 1 µl of cDNA made up to 20 µl with sterile distilled water.
The following PCR protocol was carried out in an MJ Research PTC-200 Peltier
Thermal Cycler (Bresatec, South Australia):
94°C 5 min (initial denaturation), 35
cycles at: 94°C 1 min (denaturation), 1 min annealing (temperature specified in Chapter
3), 72°C 1 min (extension) with a final extension for 8 min at 72°C. All PCR products
were electrophoresed at 100V on a 2% agarose/TBE gel containing ethidium bromide
(EtBr) and photographed under a UV lamp. PCR product size was determined by
electrophoresis with DNA molecular weight marker IX (Boehringer Mannheim) as a
guide.
2.1.3.2 Quantitative RT-PCR
β-2-microglobulin, PSA and KLK4 PCR products were excised from the gel and purified
(as described below) and the cDNA yields quantified at 260 nm using a DU 640
Beckman spectrophotometer. Standards of known cDNA concentrations were made
from each purified PCR product by 10-fold serial dilutions in Tris-EDTA pH 8.0 buffer.
5 µg of RNA extracted from hormone and growth factor treated cells were reverse
transcribed as described above.
PCR reactions were run on an LC-32 Lightcycler (Idaho Technology, Idaho Falls, Idaho,
USA). 10 µl PCR reactions were set up containing: 10X PCR buffer containing 30 mM
MgCl2 and 1 mg/ml BSA (Idaho Technology), 0.2 mM dNTPs, 100 ng/ml forward and
reverse primers, 0.5X SYBR Green I (Molecular Probes), 0.25 units of Platinum Taq
(Invitrogen) and 1 µl of template (either tissue cDNA or standard with a known copy
number). The reactions were transferred to LC capillary tubes (Idaho Technology) and
44
Chapter 2
reactions were cycled as follows: 94ºC for 2 minutes, 50 cycles of denaturation at 94ºC
for 1 second, annealing at 55-59ºC for 2 seconds (depending on primer combination as
listed Chapter 3), and extension for 20 seconds at 72°C followed by fluorescence
readings taken at 2ºC below the resultant melting temperature (84-89ºC; Recording
Temp, Chapter 3). Continuous fluorescence readings with temperature transitions of
0.2˚C/second between 72-94ºC resulted in melting curve analysis of each transcript.
The amount of β2-microglobulin, PSA and KLK4 in each cDNA sample was quantified
by direct comparison to known purified standards for each respective gene. Each cDNA
sample was quantified in triplicate in three separate PCR reactions. The PCR cycle at
which the reaction has reached its log-linear phase is determined by the Lightcycler and
this is directly proportional to the amount of starting transcript in the reaction. The
transcript copy number of a sample is calculated by comparing the cycle number
obtained for the log-linear phase of the test samples with the cycle number obtained for
the log-linear phase of known standards in the same PCR reaction.
2.1.4
Gel Purification
PCR products were visualised under a UV lamp, photographed and excised from the gel
with a sterile scalpel blade. Excised samples were purified using a Gel Extraction Kit
(QIAGEN) using the manufacturer’s protocol. Briefly, the agarose slice was dissolved
by heating in QG buffer and applied to a column that was washed, and the DNA eluted
from the column using the provided elution buffer. Following extraction, DNA
concentration was determined spectrophotometrically using a DU 640 Beckman
spectrophotometer wavelength scan analysis from 220 nm – 320 nm.
2.1.5
DNA Sequencing
PCR products were sequenced using the ABI PRISM Dye Terminator Cycle Sequencing
Ready Reaction Kit (Perkin Elmer) at the DNA sequencing facility, Australian Genome
Research Facility (University of Queensland, Brisbane, Australia).
45
Chapter 2
2.1.6
Western Blot Analysis
2.1.6.1 Intracellular Protein Extraction
Cell pellets from T80cm2 flasks were collected from tissue culture treatments. 1 ml of
cell lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1 mM PMSF,
10 mM EDTA pH 8.0, 1 tablet Roche complete protease inhibitor cocktail/25 ml) was
added to the pellet followed by drawing the cell solution through a pipette tip several
times and then 3 times through a 26-gauge needle to lyse the cells. Lysates were
centrifuged at 12000 x g for 20 min at 4°C and the supernatant aliquoted and stored at
-70°C.
2.1.6.2 Protein Quantitation
The Pierce BCA Protein Assay Kit (Progen) was used following the manufacturer’s
instructions in the 96 microwell plate format to determine protein concentration.
Increasing concentrations of BSA (0.1-1mg/ml) were used to generate a standard curve.
Samples of unknown concentration were diluted to a ratio of 1:10 in TE in a total
volume of 25 µl. Triplicate wells of each standard and sample were then incubated with
200 µl of working reagent (1:50, Part B: Part A; BCA kit) for 30 min at 37°C.
Following this incubation, the plate was cooled to room temperature and colourimetric
absorbances were determined spectrophotometrically at 550nm using the Beckman plate
reader (Beckman).
Log-log curves were generated by SoftMax software and
concentrations of each unknown sample were calculated.
2.1.6.3 Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Protein samples were separated using 10% SDS-PAGE (separating gels containing 1.5M
Tris/0.1%SDS and 40% acrylamide, overlayed with a 4% stacking gel). Protein samples
(10µg made up to a total volume of 10µl) were added to 5µl of 2x loading buffer (+/DTT) and boiled for 4 min. Samples were electrophoresed, using a Protean II minigel
apparatus (BioRad) in the Laemmli buffer system (0.0255 M Tris, 0.25 M glycine, 0.1%
w/v SDS, pH 8.3), through the stacking gel at 70mV and through the separating gel at
140 mV at 4°C. When the dye front reached the desired position, the gels were removed
46
Chapter 2
from the apparatus and placed into ice-cold 1x carbonate buffer (5 mM NaHCO3, 1.5
mM Na2CO3, pH 9.9, 20% methanol).
2.1.6.4 Western Blotting
Peptides used to generate anti-peptide hK4 antibodies were synthesised commercially
(Chiron Technologies Pty Ltd, Victoria, Australia). Antibodies were raised in rabbits
(Chiron) and affinity-purified using Sepharose 6B columns coupled with the respective
peptides (Harvey et al., 2003).
Proteins separated by SDS-PAGE were transferred to Protran nitrocellulose membrane
(Schleicher and Schuell; Medos, Brisbane, Australia) using a BioRad Transblot
apparatus. Electrophoresis was performed in 1x carbonate buffer (5 mM NaHCO3, 1.5
mM Na2CO3, pH 9.9, 20% methanol) for 2 h at 200 mA at 4°C. After protein transfer,
the membrane was stained in Ponceau Red (Sigma) for 1 min to ensure transfer and
equal loading had occurred. The membrane was rinsed in water to remove the stain and
immediately placed in 5% blocking solution (5% Skim milk powder in TBS/Tween
(0.02 M Tris-HCl, 0.5 M NaCl, pH 7.5 and 0.1% v/v Tween 20)) to block non-specific
sites for 1 h at room temperature. Primary antibodies were prepared in either 5% or
0.83% blocking solution to the required concentration and then incubated at 4°C
overnight. The blot was washed 5x10 min washes in TBS/Tween. To develop the bands
detected by the antibody, the blots were incubated in 1:5 diluted Femto substrate
(Pierce) and exposed to X-ray film (Curix Blue HC-S plus, Agfa, Brisbane, Australia)
for an appropriate period (1 min to overnight). The film was then developed using an
Agfa automatic developer (Curix).
Several blots were stripped and re-probed with other antibodies. In these cases, the
membrane was incubated in Femto stripping solution (Pierce) for 15 min at room
temperature, re-blocked in blocking solution and the incubation method repeated as
above.
47
Chapter 2
2.1.7
Immunofluorescence
Immunofluorescence analysis of cells required growing cells in 8-well or 16-well Labtek
chamber slides (Medos) in medium containing FBS until they were 80% confluent (~3
days). Each chamber was washed twice in sterile PBS followed by fixation in ice-cold
100% methanol for 5 min. The fixative was removed and the slide allowed to dry for at
least 10 min at room temperature, or until the methanol had evaporated. It was then
sealed in a plastic bag and frozen at -20°C until needed. Frozen slides were used within
one week of freezing to ensure that proteins were not degraded. When needed, slides
were defrosted using 100% methanol at room temperature for 2 min before washing in
PBS (5x3 min). The slides were blocked in 2% BSA (Fraction V, Sigma) and 0.1%
saponin in PBS for 20 min at room temperature. The primary antibody of interest was
prepared at a 1:1000 dilution in 0.1% saponin in PBS and incubated with the slide at 4°C
overnight.
The following day, the slides were washed in PBS (5x3 min) and the
appropriate secondary antibody (AlexaFluor 488, Goat anti-mouse IgG (Molecular
Probes)) was applied for 30 min. The slides were washed in PBS (5x3 min), mounted in
80% glycerol in PBS and viewed using the UV microscope (Leica Laborlux S).
48
CHAPTER THREE
THE EXPRESSION OF KLK4 IN PROSTATE CANCER
Chapter 3
3.0 INTRODUCTION
Despite the availability and use of PSA, and recently the kallikrein, hK2 (Kwiatkowski
et al., 1998, Recker et al., 1998, Partin et al., 1999, Becker et al., 2000, Nam et al.,
2000, Scorilas et al., 2003), as markers for diagnostic and prognostic use in prostate
disease management, additional markers are required to definitively differentiate benign
from malignant forms of prostate disease. As two members of the kallikrein family have
already proven useful, it was logical to search for new/adjunct biomarkers within this
family. Therefore in this study, KLK4 has been explored as a potential biomarker of
prostate disease.
At the outset of this study, little was known regarding KLK4 expression in benign and
malignant prostate tissues, although expression studies in three prostate cancer cell lines
had been performed. Expression of KLK4 was detected in the LNCaP cell line, but not in
DU145 and PC-3 cells (Nelson et al., 1999). In addition, the up-regulation of KLK4
expression levels in response to androgen stimulation in the LNCaP cell line was
demonstrated (Nelson et al., 1999).
In order to extend these studies, expression of KLK4 was examined at both the mRNA
and protein level in cell lines that represent potential models of progression from
androgen sensitive to androgen insensitive disease. These cell lines included RWPE1
and RWPE2; immortalised non-tumourigenic and tumourigenic prostate epithelial lines
respectively (Bello et al., 1997), LNCaP, an androgen dependent prostate cancer line
(Horoszewicz et al., 1983), DU145 and PC-3, both androgen independent prostate
cancer lines derived from brain and bone metastases respectively (Stone et al., 1978,
Kaighn et al., 1979). The C4 series of LNCaP sublines consists of the C4 (primary
tumour) and C4-2 (lymph node metastasis) cell lines which were derived from the
LNCaP cell line by co-culture with the human bone fibroblast cell line (MS) in male
athymic mice. The C4-2B cell line is a bone metastasis derivative from the C4-2 cell line
(Chung et al., 1997). As the C4 series of cell lines progress, they become increasingly
metastatic and androgen insensitive, thereby providing a good theoretical model to
50
Chapter 3
characterise the expression of genes, which may be important to the progression of
prostate cancer. The C4 series produce a 20-30 fold higher amount of basal steady-state
concentrations of PSA than that of the parental LNCaP cells (Thalmann et al., 2000).
The expression of KLK4 in these cell lines has not yet been characterised.
As described in Chapter 1, a remarkable feature of many members of the human
kallikrein family is the presence of alternative transcripts. Thus far, four variants in
addition to the full length KLK4 have been described (Obiezu and Diamandis, 2000,
Dong et al., 2001, Myers and Clements, 2001, Korkmaz et al., 2001). Interestingly,
results from this laboratory show an association of KLK4 transcript expression with
ovarian cancer progression (Dong et al., 2001). It was demonstrated that several KLK4
mRNA variants are expressed by ovarian tumours but not by normal ovaries. It remains
to be established whether any of these KLK4 variants may be differentially expressed
between benign and malignant prostate tissue and therefore, may be of use in providing
a more discriminating marker.
Therefore, the aims of this study were to examine the expression profile of KLK4 and its
splice variants in prostate cancer and BPH tissues, using qualitative and real-time PCR
technology, with a view to determining whether KLK4 may have potential value as a
molecular marker for prostate disease. Additionally, this study aimed to characterise the
expression profile of KLK4 and its splice variants in cell lines that represent an in vitro
model of prostate cancer progression.
51
Chapter 3
3.1 MATERIALS AND METHODS
3.1.1 Prostate Tissue Samples
Prostate tissue specimens were obtained from men who underwent either transurethral
resection of the prostate (TURP) or radical prostatectomy for prostate cancer; or TURP
or open enucleative prostatectomy for BPH, at the Royal Brisbane Hospital (RBH) or
Redcliffe Hospital, Brisbane. The collected tissues were immediately snap-frozen in
liquid nitrogen upon removal from the donor and stored at -70ºC. Each sample was
histologically assessed to confirm the pathology at the RBH by the uro-pathologist, Dr.
H.M. Samaratunga (now at Sullivan and Nicolaides Pathology Laboratories, Brisbane).
Ethics approval was obtained from the respective institutional Ethics Committees (QUT
reference numbers 0949/2H and 0949/3H; Royal Brisbane Hospital ethics number
95/88) and informed consent was obtained from all patients. A list of these samples and
relevant clinical and pathology information is shown in Table 3.1.
3.1.2 RNA Extraction and Conventional RT-PCR
Total RNA was extracted from prostate tissue specimens by Ms Tara Veveris-Lowe
(QUT) as described in Section 2.1.2. The prostate cancer cell lines, RWPE1, RWPE2,
LNCaP, DU145 and PC-3 were cultured as outlined in Section 2.1.1.2. RNA was
extracted from the frozen pellets of these cells along with the frozen pellets of the
LNCaP C4 series (C4, C4-2, C4-2B) which were supplied by Mr Daniel McCulloch
(QUT). Following RNA extraction, RT-PCR was performed as described in Section
2.1.3.1. RT-PCR with primers spanning exon 1-5 was performed for 40 cycles. Primer
sequences used in PCR reactions are listed in Table 3.2. Primers spanning exon 1 -5
were used to identify the full length KLK4 transcript. This primer set would also
amplify variants 3 and 4 if they exist as transcripts containing exon 1. Primers spanning
exon 2 – 5 were used to detect all KLK4 transcripts. Figure 3.1 illustrates the KLK4
transcripts and the position of the two primer combinations. β2-microglobulin primers
were used to identify any contaminating genomic DNA.
PCR samples were
electrophoresed on 2% agarose gels containing 0.5µg/ml ethidium bromide and selected
bands were excised for sequencing (Sections 2.1.3.1 and 2.1.5).
52
Chapter 3
Table 3.1. Surgical and pathology information for tissue preparations from 24
prostate cancer patients and 28 BPH patients
PROSTATE CANCER PATIENTS
CODE
Ca7
Ca12
Ca22
C2-99
C3-99
C9-99
RC1-99*
RC2-99*
RC3-99*
RC4-99*
RC6-99*
RC7-99*
RC8-99*
RC9-99*
RC1-00
RC2-00
RC4-00*
RC5-00
RC9-00*
RC11-00*
RC15-00*
RC18-00*
RC22-00*
RC25-00*
GLEASON
SCORE
n.a.
n.a.
n.a.
4+5
3+3
n.a.
4+3
4+5
5+5
4+3
4+3
3+4
3+4
3+3
3+3
4+5
4+3
4+4
4+5
4+3
4+3
4+5
3+4
4+5
SURGERY TYPE
n.a.
n.a.
n.a.
TURP
n.a.
TURP
RP
TURP
TURP
TURP
RP
RP
RP
RP
RP
TURP
TURP
TURP
TURP
TURP
RP
TURP
RP
RP
BPH PATIENTS
CODE
SURGERY TYPE
BPH18
TURP
BPH19
TURP
BPH21
TURP
BPH31
TURP
BPH35
TURP
B1-99
TURP
B4-99
n.a.
B5-99
TURP
B7-99
TURP
B8-99
TURP
B10-99
TURP
B11-99
OP
B12-99
OP
RB1-99*
TURP
RB2-99*
TURP
RB3-99*
TURP
RB4-99*
OP
RB1-00*
TURP
RB2-00*
TURP
RB4-00
TURP
RB5-00
TURP
RB6-00*
TURP
RB7-00*
TURP
RB8-00*
TURP
RB9-00*
TURP
RB10-00*
TURP
RB12-00
OP
RB13-00*
TURP
n.a. - Not available; OP – Open prostatectomy; RP – Radical prostatectomy; TURP - Transurethral
resection of the prostate; * - samples used for Quantitative RT-PCR
53
Chapter 3
ATG
Full Length
Exon 1
Exon 2
Exon 3
Exon 4
Exon 5
Exon 3
Exon 4
Exon 5
ATG
Exon 2
Variant 1
Exon 1 deletion
ATG
Exon 2
ATG
Exon 1
ATG
Exon 3
Exon 4
Exon 5
Variant 2
Exon 1 deletion
and Intron 3
insertion
ATG
Exon 2
Exon 3
Exon 4
Exon 5
Variant 3
Partial Intron 2
insertion
ATG
Variant 4
Exon 4 deletion
Exon 1
Exon 2
Exon 3
Exon 5
Figure 3.1 Location of RT-PCR primers for full length KLK4 and alternatively
spliced mRNA transcripts.
Full length KLK4 and four variant transcripts are illustrated. Bold black arrows indicate
the approximate position of PCR primers. The sites of two potential start codons are
indicated by the ATG. Exons are represented as purple boxes, introns as a line, and
intron insertions are represented as green boxes. Dotted lines indicate that Variants 3
and 4 may also exist as an exon 1 deleted form. The figure is not drawn to scale.
54
Chapter 3
Table 3.2: Oligonucleotide Primers for conventional RT-PCR
PCR Product
Primer sequence
Annealing
temp. (ºC)
Product
size (bp)
KLK4 ex1-5
For 5’- ATGGCCACAGCAGGAAATCCC – 3’
Rev 5’ – CAAGGCCCTGCAAGTACCCG – 3’
For 5’ – GCGGCACTGGTCATGGAAAACG – 3’
Rev 5’ – CAAGGCCCTGCAAGTACCCG – 3’
For 5’-ATCGAATTCGCACCCGGAGAGCTGTGT-3’
Rev 5’-CTGAGGGTGAACTTGCGCACAC-3’
For 5’-TGAATTGCTATGTGTCTGGGT-3’
Rev 5’-CCTCCATGATGCTGCTTACAT-3’
60
642
62
556
60
138
56
238
KLK4 ex2-5
PSA
β 2microglobulin
For = Forward primer; Rev = Reverse primer
3.1.3 Real-time Quantitative PCR of KLK4 mRNA Transcript Expression
Real-time PCR was carried out on the Idaho Technology ‘Light Cycler’ LC32 (Idaho
Technology, Inc., Utah, USA) as described in Section 2.1.3.2. Internal standard PCR
products for each mRNA transcript used in each cycle run were prepared from the
prostate cancer cell line, LNCaP, by amplifying transcript-specific products by
conventional PCR as described in Section 2.1.3.1.
Two primer sets were used to
amplify KLK4 (Table 3.3). Quantitative PCR using the Idaho Technology ‘Light Cycler’
exhibits limited efficiency in amplification when product length exceeds 300 bp.
Therefore the primer sets used for quantitative PCR differ from the conventional PCR
primers. Exon 1-3 primers were used to detect the full length and variants 3 and 4 (if
present as exon1-containing transcripts) and exon 2-3 primers were used to detect all
KLK4 transcripts.
Table 3.3 Oligonucleotide Primers for Real Time Quantitative RT-PCR
PCR
Product
KLK4
ex1-3
KLK4
ex2-3
PSA wild
type
β2-microglobulin
Primer sequence
For 5’- ATGGCCACAGCAGGAAATCCC – 3’
Rev 5`-CCCAGCCCGATGGTGTAGGAG-3`
For 5`- GCGGCACTGGTCATGGAAAAGG-3`
Rev 5`-CCCAGCCCGATGGTGTAGGAG-3`
For 5’-GCATCAGGAACAAAAGCGTGA-3’
Rev 5’-CCTGAGGAATCGATTCTTCAG-3’
For 5’-TGAATTGCTATGTGTCTGGGT-3’
Rev 5’-CCTCCATGATGCTGCTTACAT-3’
For = Forward primer; Rev = Reverse primer
55
Annealing
temp. (ºC)
59
Recording
temp. (ºC)
89
58
88
58
84
55
84
Chapter 3
A transcript-specific standard curve was generated by the LC32 software (103-1010
copies/µl). From this standard curve, DNA copy numbers for each individual tissue
cDNA were calculated. Each assay was completed three times, in triplicate, and copy
numbers were normalised to β2-microglobulin levels collected and averaged for each
individual tissue by dividing the gene copy number (ie. KLK4 or PSA) with the β2microglobulin copy number gained for each individual tissue. Normalised ratios for
each sample population were averaged and statistical analyses were performed to
determine whether the two sample groups were likely to have come from the same two
underlying populations by using a two-tailed Student’s t-test.
3.1.4 Pearson’s Correlation Analysis of Prostate Cancer Specimens
To determine the relationship between tumour grade in prostate cancer samples and the
value obtained for each transcript determined by real-time PCR, the Pearson correlation
co-efficient was calculated. The Gleason grades were added (see Table 3.1) to give the
Gleason score and plotted against each specimen’s corrected transcript value to establish
whether increasingly aggressive stages of cancer correlate to an increased expression of
particular mRNA transcripts. To make a distinction between Gleason Grade 3+4 and
4+3 (as 4+3 consists of less differentiated cancer), grade 3+4 was assigned a score of 7,
while 4+3 was assigned a score of 7.5. Calculations were performed using the Excel
program, Pearson, which returns the Pearson product moment correlation coefficient and
regression value, ‘r’.
3.1.5 Protein Extraction
Intracellular protein was extracted from cell pellets of the RWPE1, RWPE2, LNCaP,
C4, C4-2, C4-2B, DU145 and PC-3 cell lines as described in Section 2.1.6.1.
Whole
cell protein was quantitated in triplicate using the BCA protein assay reagent (Pierce,
Rockford, IL) as per the manufacturer’s ‘microwell’ protocol using Bovine Serum
Albumin (BSA) as a reference standard, as detailed in Section 2.1.6.2.
56
Chapter 3
3.1.6 Western Blot
10ug of protein from each cell line extracted was resuspended in a reducing loading
buffer and loaded onto a 10% SDS-PAGE gel, followed by standard Western blotting
procedure as detailed in Sections 2.1.6.3 and 2.1.6.4. Primary antibodies used were the
polyclonal hK4 - C terminus antibody, which detects full length and variant 1 hK4 - at a
dilution of 1:1000 (see Figure 3.2). The polyclonal Dako PSA antibody was used at a
dilution of 1:5000.
3.1.7 Immunofluorescence
The prostate cell lines RWPE-1, RWPE-2, LNCaP, DU145 and PC-3 were cultured in
standard T25 tissue culture vessels to 70% confluency and harvested with
trypsin/versine as described in Section 2.1.1.2. The cells were counted in a
haemocytometer and seeded into 16 well Labtek chamber slides (Medos) at a
concentration of 5000 cells/well and allowed to grow overnight. The precise details of
subsequent steps are outlined in Section 2.1.7.
The primary antibodies used for
detecting hK4 were the N and C terminal anti-peptide antibodies at a dilution of 1:1000.
As described above, the C terminus antibody detects full length and variant 1 hK4, while
the N terminus antibody detects full length hK4 and variants 3 and 4.
57
Chapter 3
His
Met
Pre
Pro
Ser
Asp
Full Length
Mature
N terminus Ab
Met
His
C terminus Ab
Asp
Ser
Mature
Met His
Variant 1
Asp
Mature
Variant 2
His
Met
Pre
Variant 3
Pro
His
Met
Pre
Pro
Asp
Mature
Variant 4
Figure 3.2 Position of antibodies for full length and variant hK4 proteins.
Shown are the pre-pro-protein for full length hK4 and the truncated hK4 proteins
encoded from the KLK4 splice variants. The positions of the N and C terminal peptides
from which the antibodies used in this study were raised are indicated by a green
triangle. The amino acids which comprise the catalytic triad (His/Asp/Ser) are shown
above the protein.
The figure is not drawn to scale.
58
Chapter 3
3.2 RESULTS
3.2.1 RT-PCR expression of KLK4 mRNA transcripts compared to PSA in benign
and malignant prostate tissues
All samples listed in Table 3.1 were examined twice by RT-PCR for full length KLK4,
KLK4 variants, PSA and β2-microglobulin expression. Representative gels of the RTPCR expression profiles of KLK4 and PSA mRNA transcripts in 9 prostate cancer and 9
BPH tissues are shown in Figure 3.3. The expression profile of full length KLK4 (Panel
A) is similar in both benign and malignant prostate tissue, with some but not all
expressing the full length transcript. Of interest is a slightly smaller product in lane 4 of
Panel A which was present in only one BPH sample of all tissues examined. This band
was excised for sequencing but did not yield a readable sequence. As variants 3 and 4
were not detected using the exon 1-5 primer set, it may be that these two variants exist
only as the exon 1 deleted form.
Primers spanning exon 2-5 amplify the exon 2-5 component of the full length KLK4 and
all KLK4 variants which are expressed at varying levels in both cancer and BPH samples
(Panel B). The 609 bp band is Variant 2 which is produced by an intron 3 insertion of
the exon 1 deleted form. The 526 bp band has a greater intensity and consists of two
KLK4 transcripts which are indistinguishable when using the exon 2-5 primer set. The
two transcripts are the full length KLK4 and Variant 1, which is the exon 1 deleted form.
Although difficult to see on the reproduced gel photo, Variant 4, the exon 4 deleted
form, is also present in low abundance with a faint band at 389 bp. Variant 3 (538 bp) is
not detected as it may be necessary to run the gels for a longer period of time to ensure
greater resolution of this transcript.
Panel C shows the expression profile for PSA where bands are visible in all cancer and
BPH samples. The expression profile of β2-microglobulin indicated that all samples
were of a similar quality, with no genomic contamination, giving an expected size of
238 bp (Figure 3.3, Panel D). Selected KLK4 bands were sequenced from a number of
59
Chapter 3
M
A. KLK4
exon 1 - 5
B. KLK4
exon 2 - 5
C. PSA
Cancer
BPH
872 bp
603 bp
N
642 bp
872 bp
603 bp
609 bp
526 bp
389 bp
194 bp
138 bp
118 bp
603 bp
D. β2microglobulin
234 bp
238 bp
Figure 3.3 KLK4 and PSA mRNA transcript expression profile in prostate cancer
and BPH patient tissues
Representative ethidium bromide-stained agarose gels showing the results of RT-PCR
for KLK4 mRNA transcripts and PSA in 9 prostate cancer and 9 BPH samples. β2microglobulin for these samples illustrates that no genomic contamination was present
(Panel D). Transcript sizes are indicated to the right of each panel. (Key: M – Roche
Marker IX, sizes indicated to the left of each panel; N – Negative control; bp – base
pairs).
60
Chapter 3
prostate cancer and BPH samples and confirmed as being specific to each variant
transcript (data not shown).
3.2.2 Real-time PCR analysis of KLK4 and PSA mRNA transcripts in prostate
cancer and BPH
The qualitative RT-PCR expression patterns for full length KLK4 and variant forms
revealed a profile which was essentially not different between the prostate cancer or
BPH samples. However, given the PCRs were performed for 35 cycles (exon 2-5) and
40 cycles (exon 1-5), the results are not within the linear amplification range. Other
kallikreins (PSA and KLK2) amplified under the same conditions with similar results to
those of KLK4 with conventional RT-PCR have been shown to have significant
differences
when
communication).
assessed
quantitatively
(Ms
T.
Veveris-Lowe,
personal
Therefore, real-time PCR was performed to determine absolute
transcript levels in each sample population.
A subset of tissue samples (as indicated in Table 3.1) was analysed, but insufficient
amplification of full length KLK4 prevented accurate quantitation of this transcript in all
samples (data not shown). As it is difficult to design primers to discriminate between
the variants and full length KLK4, quantitation of “total” KLK4 was performed. Primers
(exon 2-3) detecting “total” KLK4 result in a product of 109 bp which includes full
length, Variant 1 (exon 1 deleted), Variant 2 (intron 3 inclusion), Variant 3 (intron 2
inclusion) and Variant 4 (exon 4 deleted) KLK4 transcripts.
Twelve BPH samples and
16 cancer samples were assayed for KLK4. The samples used for quantitative PCR are
listed in Table 3.1 and denoted with an asterisk. Each assay was completed in duplicate
on three separate occasions for each tissue sample and each raw value obtained from the
real-time PCR was normalised to an average DNA copy number gained from assaying
β2-microglobulin quantitatively from each individual sample, and calculating a ratio
between the two genes. The mean ratio value for the pooled populations, either cancer
or BPH, was calculated for “total” KLK4 (n = 3 experiments, performed in duplicate)
and is illustrated in Figure 3.4A in graphical form.
“Total” KLK4 displayed a
statistically significant 3.5 fold greater expression in prostate cancer tissues compared to
61
Chapter 3
A
KLK4
5
*
Ratio
4
3
2
1
0
BPH
Cancer
PSA
B
5
Ratio
4
3
2
1
0
BPH
Cancer
Figure 3.4 Real-time PCR analysis of KLK4 mRNA in prostate cancer and BPH
Graphical representation of real-time PCR analysis of cancer samples and BPH samples
for “total” KLK4 and wild type PSA (12 BPH, 16 cancer samples). Quantitative PCR
for each tissue sample was performed in triplicate in three separate PCR assays. Copy
numbers were normalised to β2-microglobulin levels collected and averaged for each
individual tissue by dividing the gene copy number (ie. KLK4 or PSA) with the β2microglobulin copy number gained for each individual tissue. Normalised ratios for
each sample population were averaged and statistical analyses were performed.
Statistical significance (indicated with an asterisk) for “total” KLK4 was assessed using
the Student’s t-test with p<0.05.
62
Chapter 3
the benign population (p = 0.04).
Quantitative PCR and subsequent statistical analysis for wild type PSA was performed
by Ms Tara Veveris-Lowe using the same method as for KLK4 quantitation. The PSA
data was included in order to compare to the KLK4 quantitative data, as the same
individual tissue samples were assayed. Figure 3.4 B shows a 1.4 fold increase in PSA
transcript level in the prostate cancer tissues compared to the benign tissues. This
increase was not statistically significant (p = 0.15).
3.2.3 Correlation of tumour grade (Gleason score) to transcript level
Calculation of the correlation co-efficient between the Gleason score for prostate cancer
specimens and transcript ratio for “total” KLK4 variant showed no correlation (r =
0.075) (Figure 3.5).
Regression co-efficients calculated for wild type PSA also
demonstrated no association between grade and mRNA levels (r = 0.13).
3.2.4 RT-PCR expression of KLK4 and PSA mRNA transcripts in prostate cancer
cell lines
Figure 3.6 shows the RT-PCR expression profile of KLK4 across a range of cell lines.
Full length KLK4 (exon 1 - 5 amplification) is only expressed in the LNCaP cell line and
the C4 series, which were derived from the LNCaP line (Panel A).
As previously
shown for the exon 1-5 RT-PCR in the tissue samples (Figure 3.3A), the one product
corresponding to full length KLK4 indicates that variants 3 and 4 exist only in the exon 1
deleted form in these cell lines. Although only a qualitative assessment, there is only
very low expression of the full length transcript, even at 40 PCR cycles. All other PCR
reactions were performed for 35 cycles. Panel B displays the results of the exon 2- 5
PCR with four KLK4 bands present in the LNCaP, DU145 and PC-3 cell lines. The 609
bp band is Variant 2, the 526 bp band consists of full length KLK4 and Variant 1, which
are indistinguishable when using the exon 2-5 primer set; and the 389 bp band is Variant
4. Variant 3 (538 bp) is not detected, as it may be necessary to run the gels for a longer
period of time to ensure greater resolution of this transcript. An additional band is seen
63
Chapter 3
KLK4
T ran scrip t R atio
40
30
20
10
0
5
6
7
8
9
10
11
Gleason Score
PSA
3
Transcript Ratio
2.5
2
1.5
1
0.5
0
5
6
7
8
9
10
11
Gleason
Score
Gleason Score
Figure 3.5 Correlation of tumour grade to transcript type
Scatter plots indicating the relationship between tumour grade (Gleason score 6-10) and
transcript ratio (compared with β2-microglobulin) for “total” KLK4 and PSA mRNA (n
= 16 cancer samples). The regression line is drawn in green. Pearson’s correlation coefficient for KLK4: r = 0.075, indicates that no correlation was found between tumour
grade and transcript ratio. Pearson’s correlation co-efficient for PSA: r = 0.13.
64
Chapter 3
M
A. KLK4
exon 1 - 5
R1
R2
L
D
P
M
N
872 bp
603 bp
872 bp
B. KLK4 603 bp
exon 2 - 5
118 bp
310 bp
D. β2microglobulin
194 bp
C4
C4-2 C4-2B N
872 bp
642 bp
603 bp
642 bp
←609 bp
←526 bp
←389 bp
←609 bp
←526 bp
←389 bp
194 bp
194 bp
C. PSA
L
138 bp
118 bp
138 bp
238 bp
281 bp
234 bp
238 bp
Figure 3.6 KLK4 and PSA mRNA transcript expression profile in prostate cell lines
Ethidium bromide-stained agarose gels showing the results of RT-PCR for full length
KLK4 (exon 1 - 5, Panel A), all KLK4 transcripts (exon 2 – 5, Panel B) and PSA (Panel
C) mRNA transcripts in a range of prostate cell lines representing normal epithelial cells
to androgen insensitive disease.
β2-microglobulin amplification for these samples
illustrates that no genomic contamination was present (Panel D). Transcript sizes are
indicated to the right of each panel, Molecular weight marker sizes indicated to the left
of each panel. Cell lines on the gels in the left hand panel: R1 – RWPE1; R2 – RWPE2;
L – LNCaP; D – DU145; P – PC-3; N – Negative control. Cell lines on the gels in the
right hand panel: L – LNCaP; C4 subline; C4-2 subline; C4-2B subline. N – Negative
control; M – Roche Marker IX; bp – base pairs.
65
Chapter 3
at approximately 510 bp.
This band was excised and sequenced, but a readable
sequence was not obtained. It is most likely that this band corresponds to a partial exon
deletion of either the full length or Variant 1 transcript. On the reproduced gel photo of
the C4 series PCR (Figure 3.6, Panel B, right hand gel), it is difficult to see all four
bands, as it was not electrophoresed for the same length of time as the left hand gel in
Panel B.
It is interesting to note the low expression of only one 526 bp band
(corresponding to the full length/exon 1 deleted transcripts) in the RWPE2 cell line, and
the lack of expression of all KLK4 transcripts in the non-tumourigenic RWPE1 cell line.
PSA expression was also assessed and compared to the known expression profile. As
expected, PSA was only detected in the LNCaP and C4 series. The expression profile of
β2-microglobulin shows that all samples were of a similar quality, with no genomic
contamination, giving an expected size of 238 bp (Figure 3.6, Panel D). These results
were found in three separate RNA preparations of each cell line that were amplified on
different occasions.
3.2.5 The expression of hK4 in prostate cancer cell lines
Western blot analysis shows qualitative expression profiles of hK4 and PSA in RWPE1,
RWPE2, LNCaP, DU145, PC-3, C4, C4-2 and C4-2B cell protein extracts (Figure 3.7).
Using an anti-C-terminus peptide antibody, hK4 was detected in all but the RWPE1 cell
line with two bands at approximately 40 KDa (Panels A and D). As indicated in Figure
3.2, the anti-C-terminus antibody would recognise hK4 protein encoded from full length
and Variant 1 KLK4 transcripts and it is likely to be these two proteins which are
detected on the blots. PSA protein expression revealed the expected profile with a
predominant 33 KDa band in only the LNCaP and C4 series cell lines (Thalmann et al.,
2000)(Panels B and E). The housekeeping gene β-tubulin was detected on the same
membrane to determine protein loading variability between lanes (Panels C and F). The
size of each band was determined by comparison with Biorad pre-stained protein marker
run on the same PAGE gel (Figure 3.7). These results were found in three separate
whole cell protein lysate preparations of each cell line analysed on three separate
occasions.
66
Chapter 3
R1
A. hK4
B. PSA
R2
L
D
P
52.9
40 KDa
35.4
38 KDa
35.4
33 KDa
92
C. β- Tubulin
LNCaP
D. hK4
54 KDa
52.9
C4
F. βTubulin
C4-2b
40 KDa
52.9
38 KDa
35.4
E. PSA
C4-2
35.4
33 KDa
92
54 KDa
52.9
Figure 3.7 hK4 and PSA protein expression profile in prostate cell lines
Western blot analysis of hK4 (A and D) and PSA (B and E) protein in a range of
prostate cell lines representing a spectrum of normal epithelium to malignant disease. βTubulin blots (C and F) illustrate protein loading variability between sample wells.
Biorad marker sizes are indicated to the left of each panel and protein sizes are to the
right. (Key: R1 – RWPE1; R2 – RWPE2; L – LNCaP; D – DU145; P – PC-3).
67
Chapter 3
3.2.6 Immunofluorescence
Figure 3.8 shows photomicrographs of hK4 protein expression in the prostate cell lines
RWPE1, RWPE2, LNCaP, DU145 and PC-3 as detected using the anti-C-terminus
peptide antibody. Immunofluorescent staining for hK4 using this antibody is likely to
detect full length and Variant 1 protein products (Figure 3.2). hK4 is present in the
RWPE2, LNCaP, DU145 and PC-3 cells, corresponding to both the RT-PCR and
Western blot profiles. In the LNCaP cells, hK4 was predominantly cytoplasmic,
however in the RWPE2, DU145 and PC-3 cells the staining was weak and appeared to
have a perinuclear distribution. The negative control and the RWPE1 cells show
negligible staining.
Figure 3.9 shows immunofluorescent staining of hK4 protein in the same cells as above,
but detected with the anti N terminus peptide antibody. This antibody is likely to detect
the full length hK4 protein and, if translated, variants 3 and 4, as indicated in Figure 3.2.
A similar staining pattern is observed as for the C terminus antibody. Again, hK4 is
present in the RWPE2, LNCaP, DU145 and PC-3 cells, corresponding to both the RTPCR and Western blot profiles. As full length KLK4 is detected at the mRNA level in
only the LNCaP cell line, it is possible that the weak staining observed in the RWPE2,
DU145 and PC-3 cells is attributed to the variant 3 and 4 proteins. The staining in the
cells appeared to be perinuclear for these cells, although the LNCaP cells had a primarily
cytoplasmic distribution. Negligible staining was observed in the negative control (no
primary antibody added) and the RWPE1 cells.
68
Chapter 3
A.
B.
C.
D.
E.
F.
Figure 3.8 Immunofluorescence analysis for hK4 expression in prostate cell lines
using the C terminus Ab
Immuno-staining is indicated by the green Alexa-Fluor stain. Staining is most abundant
in the LNCaP cell line followed by RWPE2, PC-3 and DU145. Key: A. RWPE1; B:
RWPE2; C: LNCaP; D: DU145; E: PC-3; F: Negative control (no primary
antibody).
69
Chapter 3
A.
B.
C.
D.
E.
F.
Figure 3.9 Immunofluorescence analysis for hK4 expression in prostate cell lines
using the N terminus Ab
Immuno-staining is indicated by the green Alexa-Fluor stain. Staining is most abundant
in the LNCaP cell line followed by RWPE2, PC-3 and DU145. Key: A. RWPE1; B:
RWPE2; C: LNCaP; D: DU145; E: PC-3; F: Negative control (no primary
antibody).
70
Chapter 3
3.3 DISCUSSION
Diagnosis of prostate cancer is based on the suspected asymmetry of the gland detected
by digital rectal examination (DRE), serum PSA levels and subsequent biopsy. DRE is a
long-established test used by physicians to detect palpable changes in the prostate gland.
However the test can only detect cancers that have become nodular and therefore
microscopic cancers are often missed.
Abnormal cellular growth, characteristic of
cancer, disrupts the prostatic architecture allowing PSA to be released into the
circulation at high concentrations, providing the basis for the PSA serum test (Lalani et
al., 1997; Barry, 2001). Although PSA is currently the most useful marker for early
detection of prostate cancer, it does not specifically discriminate between prostate cancer
and BPH (Becker et al., 1997; Rosalki & Rutherford, 2000). Additionally, the PSA test
cannot distinguish between slow growing/latent disease and aggressive/metastatic
cancer. Furthermore, the use of PSA as a screening test has led to concern about the high
numbers of false positive results, the distress caused by a false cancer diagnosis and the
effect on patients of the need to undergo further invasive tests such as transrectal
ultrasound (TRUS) and biopsy (Gray, 2001). Considering these shortcomings, a great
deal of research is directed towards finding a more specific marker of prostate cancer.
The findings of this chapter have shown that KLK4 may have some potential as a
biomarker for prostate disease.
Although qualitative PCR analysis of full length KLK4 and a number of KLK4 variant
transcripts showed no obvious difference in expression patterns between benign and
malignant prostatic tissue, quantitative analysis revealed otherwise. Real-time PCR
analysis shows that “total” KLK4 mRNA levels are expressed to a higher degree in
prostate cancer tissues than benign samples. Statistical analysis revealed a significant
difference (P<0.05) with a 3.5-fold increase in the KLK4 level in cancer tissues
compared with BPH tissues. The quantitative analysis of PSA transcript levels in the
same tissue samples indicated a 1.4 fold increase which was not significant (P = 0.15)
due to large standard errors. Given the ambiguity of the PSA test in the “grey zone”
where high PSA serum levels can be found in men with BPH, this differential expression
71
Chapter 3
of “total” KLK4 has potential clinical value for RT-PCR analysis of biopsy tissues,
circulating cancer cells or shed cancer cells in urine and/or ejaculate as a more
discriminating marker of prostate disease. However, in order to further confirm the
potential clinical value of “total” KLK4, more extensive studies involving a larger
patient cohort are needed. Additionally, while full length KLK4 itself (independent of
other variants) was unable to be accurately quantitated in this study, it may prove useful
to pursue this further, perhaps with a more sensitive and advanced model Light cycler.
Additionally, it would also be worthwhile to discriminate between each variant to
determine which might be the most important.
While this study demonstrated differential expression of KLK4 between benign and
malignant prostate tissue, other reports are inconsistent with this finding. Day and
colleagues (2002) found that KLK4 mRNA had elevated levels in all normal, benign and
tumour samples examined. However, only 3 normal and 3 BPH samples were compared
to 24 prostate tumour samples. Therefore, the Day study (2002) also requires a larger
patient cohort to confirm their results before a significant conclusion can be reached. It
is important to note that, although not stated, the primers used in their study also
detected full length KLK4 along with each KLK4 variant transcript indicated in Figure
3.1.
As the potential use of an RT-PCR approach to disease diagnosis may not be useful on a
routine basis, it would be of interest to examine the protein expression levels of hK4 in
benign and malignant prostates. Immunohistochemical analysis of tissue sections within
this laboratory has demonstrated that hK4 is expressed to a greater degree in prostate
cancer in comparison to BPH sections (Ms L Bui, personal communication) confirming
the results documented here at the mRNA level. However, in a study by Obiezu and
colleagues (2002) it was found that hK4 concentrations were highest in healthy prostate
extracts compared with cancerous extracts, using a sandwich-type immunoassay (Obiezu
et al., 2002). The authors concluded that although KLK4 mRNA is readily detectable by
RT-PCR, hK4 protein is present in some prostatic tissue extracts but at relatively low
concentrations and that the protein is either not synthesised efficiently or is degraded
72
Chapter 3
quickly. Another explanation for the low protein concentrations is the possibility that
not all the KLK4 variants are being translated. In fact, of the four mRNA variants, only
one variant (Korkmaz/exon 1 deleted) would translate into a protein with functional
serine protease activity.
Therefore, the low hK4 abundance detected in the
immunoassay may reflect the levels of only the full length and the exon 1 deleted hK4
proteins. Additionally, the antibodies used in the immunoassay were raised against
secreted recombinant hK4 and therefore may only detect full length hK4.
The
immunohistochemical data generated by Ms L Bui was performed using an antibody
raised against an hK4 N terminus peptide which would detect hK4 proteins translated
from both full length and Variants 3 and 4, assuming all are translated.
Using the Pearson’s correlation co-efficient, it was found that there was no correlation
between tumour grade and transcript level in the tissue samples studied. As only 16
cancer samples were analysed, a larger cohort with greater representation across cancer
grades is required for a definitive outcome to be established. In order to gain further
information as to whether KLK4/hK4 may be expressed temporally or differentially with
progression, we examined the mRNA expression profile of KLK4 and its variants in two
“normal” epithelial prostate cell lines (RWPE 1 and RWPE 2), three well established
prostate cancer cell lines representing androgen dependent prostate tumours (LNCaP)
and androgen independent tumours (DU145 and PC-3) and an in vitro metastatic cell
line model (the C4 series), derived from the prostate cancer cell line LNCaP.
Whilst full length KLK4 mRNA expression was detected in the LNCaP cell line and the
progressively metastatic LNCaP C4 sublines, the KLK4 variant transcripts were detected
in all but the RWPE1 cell line. It is interesting to note that only one band was present in
the RWPE2 cell line. This 529 bp band corresponds to two products: the full length and
the exon 1 deleted variant which cannot be distinguished by size using these primers.
Therefore in the normal epithelial cell lines, the KLK4 variants are either expressed at a
low level, or not at all. However it is necessary to point out that the RWPE1 cell line,
although derived from normal prostate tissue, now consists of greater than 50
chromosomes as a result of the cell immortalisation process (Bello et al., 1997).
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Chapter 3
Despite this, RWPE1, in stark contrast to PC-3, is non-tumorigenic when seeded into
nude mouse models (Bello et al., 1997, Kim et al., 2003). Hence, for the purpose of
these expression studies, the RWPE1 cell line is least tumourigenic and the closest cell
line available resembling normal prostate epithelium. The lack of expression of KLK4
and its variants in the “normal” prostate cell line (RWPE1) compared to the cancer cell
lines is similar to that observed in ovarian cancer samples where the same three KLK4
variants were detected in different ovarian tumours but not in normal ovaries (Dong et
al., 2001). Additionally, the level of KLK4 expression was higher in late stage ovarian
tumours than in normal ovaries, indicating that the level of KLK4 and KLK4 variants is
associated with ovarian cancer progression (Dong et al., 2001).
Variant mRNA
transcripts are a common feature of the human KLK family and have been demonstrated
for other members including KLK1 (Rae et al., 1999), KLK2 (Rittenhouse et al., 1998),
KLK3 (Heuze et al., 1999, Tanaka et al., 2000, Heuze-Vourc'h et al., 2003), and KLK13
(Yousef et al., 2000). However it is important to consider that the KLK4 variants 2, 3
and 4 have premature stop codons that would lead to C-terminally truncated hK4
proteins if translated. The proposed protein products of these variants would not contain
Ser207 of the catalytic triad and are therefore unlikely to encode proteins with serine
protease activity. Nonetheless, it is necessary to determine whether any of these variant
forms could be useful diagnostic or prognostic markers for monitoring of prostate
disease.
The hK4 expression profile in the cell lines confirmed the RT-PCR analysis, with hK4
expression found in all cell lines examined except RWPE1. No obvious changes in
levels of hK4, when compared to the levels of the housekeeping gene, β-tubulin, were
observed. Two bands were distinguished at approximately 38 KDa and 40 KDa. The
predicted molecular weight of hK4 is ∼ 30 KDa and it has been suggested that the
difference in size may be due to post-translational modification as the predicted hK4
amino acid sequence contains N-glycosylation sites (Dong et al., 2001). It is likely that
the two proteins detected with the C-terminus anti-peptide antibody correspond to the
full length KLK4 mRNA transcript and Variant 1 (the exon 1 deleted transcript), as the
74
Chapter 3
other variants are predicted to produce truncated proteins which the antibody would not
recognise.
To extend these observations, we explored the localisation of hK4 within the cell using
immunofluorescence. Again, hK4 was detected in RWPE2, LNCaP, DU145 and PC-3
cells, but not RWPE1. With the C terminus antibody the greatest amount of staining
was observed in the LNCaP cells with a predominant cytoplasmic localisation,
indicative of the full length hK4. Less hK4 was observed in the RWPE2, DU145 and
PC-3 cells but with a distinct perinuclear staining pattern. Since the RT-PCR using
primers which span exon 1 -5 detected full length KLK4 in only the LNCaP cell lines, it
is likely that the weak perinuclear staining detected by the C terminus antibody in the
RWPE2, DU145 and PC-3 cells is attributed to the variant 1 protein.
Previously
published data suggest that the exon 1 deleted KLK4 mRNA variant (variant 1) is not
secreted and remains intracellular with a perinuclear localisation (Korkmaz et al., 2001).
A similar staining pattern was observed in the cells when an N terminus anti-peptide
antibody was used. Since this antibody recognises full length hK4 and variants 3 and 4,
it is likely that the weak perinuclear staining observed in the RWPE2, DU145 and PC-3
cells is due to the presence of variants 3 and 4, based on the RT-PCR results which
detect full length KLK4 in only the LNCaP cell line. Therefore, the cytoplasmic staining
observed in the LNCaP cells is due to the presence of the full length hK4. Interestingly,
immunohistochemical localisation data from the ovarian cancer studies revealed focal
membrane localisation of hK4 in addition to cytoplasmic staining in the tumour tissues.
Due to five predicted myristoylation sites in the hK4 sequence, a cell membrane
function for hK4 is also possible (Dong et al., 2001). Additional information regarding
the cellular distribution of hK4 would have benefited from co-staining for other
cytoplasmic structures. Clearly additional studies are necessary to clarify the complex
hK4 expression and localisation data to determine if there are cancer specific differences
in order to fully establish whether hK4 may be useful as a marker of prostate disease.
The results of this study have demonstrated the potential usefulness of “total” KLK4 as a
biomarker of prostate disease due to the differential expression pattern observed between
75
Chapter 3
benign and malignant prostate tissues. It has also further characterised the expression
profile of hK4/KLK4 and its mRNA variants in a range of cell lines representing normal
to malignant disease.
Further investigation is required in order to elucidate the
usefulness of KLK4/hK4 and the variants in diagnosis and/or prognosis associated with
prostate cancer progression.
76
CHAPTER FOUR
THE REGULATION OF KLK4
IN THE PROSTATE CANCER CELL LINE, LNCAP
Chapter 4
4.0 INTRODUCTION
The prostate gland relies on a variety of hormones and growth factors for its normal
growth and development, many of which are implicated in the growth and maintenance
of prostate malignancies. While the role of the male sex hormone, androgen, in normal
prostate homeostasis and prostate cancer is important, alone it is insufficient to maintain
prostate cell survival.
The IGF family, EGF, TGF-α, FGF, thyroid hormone and
endothelial growth factors are the main stimulatory regulators of proliferation in the
prostate, while the TGF-β family is the main inhibitory regulator. These growth factors
exert autocrine and paracrine effects upon stromal and epithelial cells and, with other
factors and binding proteins, control prostate cell growth (Russell et al., 1998).
The role of androgens, namely DHT, in the prostate has long been known to be of
primary importance to the development and maintenance of normal prostatic structure
and function (Bentel and Tilley, 1996). So much so that, upon removal of the androgen
supply, the prostate undergoes atrophy and involution as a direct result of epithelial cell
apoptosis ( McConnell, 1990, McConnell, 1995, Montalvo et al., 2000). In the malignant
prostate, androgens increase the transcription of a number of mitogenic growth factors in
epithelial and stromal cells which can act in an autocrine and/or paracrine manner on the
epithelium to regulate cell growth, differentiation and apoptosis (Farnsworth, 1999).
Additionally, DHT up-regulates the transcription of specific genes associated with
prostate cancer, including the kallikreins, PSA and KLK2 (Murtha et al., 1993).
Another important stimulator of cellular growth in the prostate is triiodothyronine (T3).
T3 has been shown to induce a proliferative response in LNCaP cells (Esquenet et al.,
1995) and has been defined as one of the most critical components to support growth of
LNCaP cells in serum-free defined medium (Hedlund and Miller, 1994). Recent studies
have demonstrated the interactive effects of T3 and androgens on prostate cell growth
and gene expression (Zhang et al., 1999). This study reported that T3, in the absence of
androgens, repressed the expression of KLK2, while androgens, T3 or a combination of
the two, produced a dose dependent up-regulation of PSA.
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Chapter 4
Also linked to prostate cancer development is EGF. Increased expression of EGF and
the EGF receptor in prostate cancers in comparison with benign tissue has been observed
(Harper et al., 1993; Glynne-Jones et al., 1996; Olapade-Olaopa et al., 2000).
Furthermore, their expression has been associated with prostate cancer cells undergoing
androgen independent progression (Schuurmans et al., 1989;
Chung et al., 1992).
Studies utilising PC-3 cells in a Boyden chamber microinvasion assay indicate that EGF
enhances prostate tumour cell invasion (Jarrad et al., 1994). Thus, this growth factor
represents a key modulator of prostate cancer progression.
In order to characterise the growth factor responsiveness of genes, which may play key
roles in prostate cancer progression, regulation studies are commonly carried out in
prostate cancer cell lines. The LNCaP cell line has been extensively used to study the
regulation of many genes, including those of the kallikrein family. Both PSA and KLK2
have been shown to be up- and down-regulated by a variety of growth factors and
hormones in the LNCaP cell line (Young et al., 1992, Hsieh et al., 1996, Thalmann et
al., 1996, Cleutjens et al., 1997, Hsieh et al., 1997, Hedlund et al., 1997, Sica et al.,
1999, Zhang et al., 1999, Lin et al., 2000, Mitchell et al., 2000b, Davis et al., 2002). It
is possible that, like PSA and KLK2, KLK4 may also be regulated in a similar fashion.
The previous chapter in this thesis detailed the expression of full length and variant
KLK4 transcripts in benign and malignant prostate tissues and a range of prostate cancer
cell lines. As the full length transcript (in addition to the variants) is only expressed in
the LNCaP cell line, this cell line was chosen as a good model to study the regulation of
this gene in prostate cancer.
At the outset of this study, only a few reports had detailed KLK4 regulation in hormone
dependent cancers. Expression of KLK4 was known to be up-regulated by androgens in
prostate (Nelson et al., 1999) and breast cancer cells (Yousef et al., 1999). Additionally,
progestins were found to regulate KLK4 expression in the breast cancer cell line BT-474
(Yousef et al., 1999). Given these early findings, the responsiveness of PSA and KLK2
to various hormones and growth factors in the prostate, and the fact that growth factors
are known to play an important role in prostate tumourigenesis, it was important to
79
Chapter 4
further characterise the hormone and growth factor regulation of KLK4 in prostate
cancer. In this study, DHT, T3 and EGF have been examined as these hormones/growth
factors are involved in the regulation of cancer progression and the stimulation of genes
important in prostate cancer, including the kallikreins PSA and KLK2.
Thus, the aims of this chapter were to assess the responsiveness of KLK4 mRNA and
protein levels to DHT, DHT plus T3 and T3 alone. Additionally KLK4 mRNA and
protein levels were assessed for their responsiveness to increasing concentrations of
EGF in the LNCaP cell line. PSA mRNA and protein levels were used as a control given
the previous reported responses of PSA to these factors. This chapter will build on
previously published regulation studies of KLK4 and provide evidence for the hormone
responsiveness of KLK4.
80
Chapter 4
4.1 MATERIALS AND METHODS
4.1.1 Cell Culture
The LNCaP cell line was cultured to 70% confluency in T80 culture flasks as described
in Section 2.1.1. The cells were transferred into serum-free (SF) RPMI-1640/ 0.01%
BSA/Penicillin/Streptomycin for 24 h prior to the onset of regulation studies. Test
medium was added to the cells following a warm wash (37°) with PBS. Androgen
regulation was tested using 5α-dihydrotestosterone (DHT) (Sigma, St Louis, MO, USA)
at concentrations of 0 nM (control) and 10 nM in the presence or absence of 100 nM
triiodothyronine (T3) (Sigma). For EGF regulation, human recombinant EGF (Gropep,
Adelaide) was added at concentrations of 0 (control), 10, 50 and 100 ng/ml. The cells
were cultured for a further 48 h under test conditions. At 24 h steroid or growth factor
levels were replenished by a change of fresh medium. Cells were harvested as described
in Section 2.1.1 and pellets stored at -80°C. The experiments were carried out on three
separate occasions. The EGF experiments were conducted by Mr Daniel McCulloch,
and RNA and protein generated from these treatments was used for subsequent analysis.
4.1.2 PSA Assay
To determine the biological effectiveness of the treatments, test media were analysed by
the “Automated Chemiluminescence System” for PSA (ACS:180 E-PSA) by Mr Greg
Ward (Clinical Biochemistry Department, Princess Alexandra Hospital (PAH),
Annerley, QLD, Australia). The E-PSA assay is routinely used clinically for the
detection of prostate disease. Briefly, conditioned medium was removed from the cell
monolayer, prior to collection of the cell pellet, and transferred to a clean tube. The
medium was centrifuged to remove cell debris at 1000 g for 5 min. 300 µl of
conditioned medium was assayed for PSA immunoreactivity. The assay principle relies
on a 2-site sandwich immunoassay with 2 antibodies [one PSA monoclonal antibody
(mAb) and one PSA polyclonal antibody (pAb)] with the concentration of PSA present
in the samples being relative to the chemiluminescence detected by the system. PSA
81
Chapter 4
levels measured were normalised to total protein as determined by the Pierce BCA assay
kit (Progen; see Section 2.1.6.2).
4.1.3 RNA Extraction and RT-PCR
Total RNA was extracted from cell pellets as described in Section 2.1.2. Following
RNA extraction, reverse transcription was performed as described in Section 2.1.3.1 to
generate cDNA. All cDNAs were screened for genomic DNA contamination with β2microgloblin primers, which span an intron, and were found to be free of gDNA
contamination.
4.1.4 Real-time Quantitative PCR of KLK4 mRNA Transcript Expression
Real-time PCR was carried out on the Idaho Technology ‘Light Cycler’ LC32 (Idaho
Technology, Inc., Utah, USA) as described in Section 2.1.3.2. Internal standard PCR
products for each mRNA transcript used in each cycle run were prepared from the
prostate cancer cell line, LNCaP, by amplifying transcript-specific products by
conventional PCR as described in Section 2.1.3.1. PCR products were purified using the
Qiagen gel extraction kit and the DNA copy number was calculated before serially
diluting them for use in a standard curve (103-1010 copies/µl). As it is not possible to
design primers to discriminate between full length KLK4 and the variants, quantitation
of “total” KLK4 was performed. Primers (exon 2-3) detecting “total” KLK4 result in a
product of 109 bp which includes full length, Variant 1 (exon 1 deleted), Variant 2
(intron 3 inclusion), Variant 3 (intron 2 inclusion) and Variant 4 (exon 4 deleted) KLK4
transcripts (Figure 3.2).
Real-time quantitative RT-PCR was performed for β2-
microglobulin, PSA and “total” KLK4. Primer sequences are detailed in Table 3.3.
Quantitative PCR was performed in triplicate in three separate PCR runs for three
separate experiments for each hormone/growth factor treatment and copy numbers were
normalised to corresponding β2-microglobulin levels.
4.1.5 Protein Extraction
Intracellular protein was extracted from cell pellets as described in Section 2.1.6.1.
Whole cell protein was quantitated in triplicate using the BCA protein assay reagent
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Chapter 4
(Pierce, Rockford, IL) as per the manufacturer’s ‘microwell’ protocol using Bovine
Serum Albumin (BSA) as a reference standard as detailed in Section 2.1.6.2.
4.1.6 Western Blot
10ug of protein was suspended in a reducing loading buffer and loaded onto a 10% SDSPAGE gel, followed by standard Western blotting procedure as detailed in Sections
2.1.6.3 and 2.1.6.4.
Western blots were performed twice from three separate
experiments for each growth factor tested. Primary antibodies used were the polyclonal
hK4 - C terminus antibody, which detects full length and variant 1 hK4, at a dilution of
1:1000, and the polyclonal Dako PSA antibody at a dilution of 1:5000.
4.1.7 Quantitation of signal intensity
Densitometric analysis of Western blots was carried out using a GS-690 image
densitometer (Biorad). Western blots were performed on protein samples collected from
three separate experiments (n=3). For each set of treatments, Western blots were
performed in duplicate on which subsequent densitometric analysis was performed. The
signal intensity obtained for hK4 and PSA expression was normalised for the signal
obtained for β-tubulin on the same Western blot. Absolute ratios of intensity between
control and test concentrations were calculated and the entire data set was subjected to a
one-way ANOVA, followed by a Tukey Post-Hoc analysis.
83
Chapter 4
4.2 RESULTS
4.2.1
Regulation of PSA and KLK4 in LNCaP cells by DHT and T3
To assess the effect of DHT and T3 on PSA and KLK4 mRNA and protein levels, three
individual cell culture experiments were performed. Gene and protein expression was
determined by immunoassay (for PSA), quantitative RT-PCR and Western blot.
4.2.1.1 PSA assay of conditioned medium
To determine the biological effectiveness of the treatment, PSA protein levels which are
known to be stimulated by DHT in a dose dependent manner (Montgomery et al., 1992;
Lee et al., 1995), were determined. Figure 4.1 displays the combined results of 3
experiments. As expected, PSA secreted into the medium significantly increased
following the addition of 10 nM DHT with a 13 fold increase over the control. A further
increase in PSA secretion (up to 20 fold) was observed for 10 nM DHT in combination
with 100 nM T3, while no significant increase in PSA secretion (1.5 fold) was observed
for 100 nM T3 alone. The levels of PSA secreted in response to the combined treatment
of DHT and T3 was greater than when treated with DHT alone suggesting a possible
synergistic effect, however statistical significance was not obtained between these two
treatments due to considerable inter-assay variation. Nonetheless, the significant upregulation of PSA secretion in response to DHT compared to the untreated control
demonstrates that the LNCaP cells were responsive to DHT under the in vitro cell
culture conditions used in this study.
4.2.1.2 PSA and KLK4 mRNA regulation by DHT and T3
To determine whether PSA and KLK4 are regulated at the mRNA level by DHT or T3,
quantitative PCR was performed on cDNA reverse transcribed from total RNA extracted
from LNCaP cells treated with DHT, DHT plus T3 or T3 alone. Treatment with DHT and
DHT in combination with T3 significantly up-regulated PSA and KLK4 mRNA
transcripts when compared to the untreated control (Figure 4.2). PSA was the most
responsive with a 5 fold increase in response to DHT alone and a 6 fold increase in
84
Chapter 4
PSA Protein Secretion
*
Foldng/ml
Change
25
20
*
15
10
5
0
Ctrl
DHT
DHT/T3
T3
Figure 4.1 The regulation of secreted PSA protein by DHT and T3 in LNCaP cells
LNCaP cells were untreated (control/Ctrl), or treated with 10 nM DHT, 10 nM DHT
plus 100 nM T3 or 100 nM T3 alone and cultured for 48 h. To determine the biological
effectiveness of the treatements, a PSA immunoassay was performed on conditioned
medium from three independent experiments. Data are presented as the fold increase
over 48 h untreated control (Ctrl) LNCaP cells (assigned a value of 1), with standard
error of the mean indicated by the vertical bars and statistical significance assessed using
a one-way ANOVA followed by Tukey Post-Hoc analysis (n=6; p<0.001).
85
Chapter 4
A
PSA mRNA
**
8
Fold Increase
7
**
6
5
4
3
2
1
0
Ctrl
B
DHT
DHT/T3
T3
KLK4 mRNA
6
***
5
4
*
3
2
1
0
Ctrl
DHT
DHT/T3
T3
Figure 4.2 The regulation of PSA and KLK4 mRNA by DHT and T3
Quantitative PCR analysis of DHT and T3 treated LNCaP cells assayed for PSA and
“total” KLK4 mRNAs. Data are presented as the fold increase over 48 h untreated
control (Ctrl) LNCaP cells (assigned a value of 1), with standard error of the mean
indicated by error bars. Quantitative PCR was performed in triplicate in three separate
PCR runs for three separate experiments for each hormone/growth factor treatment.
Statistical significance was assessed using a one-way ANOVA followed by Tukey PostHoc analysis (*P<0.05; **P<0.01; ***P<0.001).
86
Chapter 4
response to DHT in combination with T3, while KLK4 exhibited a 2.6 and 4.2 fold
increase for the respective treatments. While the levels of PSA and KLK4 mRNA
expressed in response to the combined treatment of DHT and T3 was greater than when
treated with DHT alone suggesting a potential synergistic effect, statistical significance
was not obtained between these two treatments. Neither PSA nor KLK4 showed a
significant change in response to T3 treatment alone. The PSA mRNA data parallels the
finding for the secreted PSA protein as described above. In addition, these data suggest
that KLK4 is regulated in a similar manner to PSA in response to these two stimulatory
factors.
4.2.1.3 PSA and hK4 protein expression in response to DHT and T3 treatment
Once it had been established that both PSA and KLK4 transcripts were regulated by
DHT and T3, Western blot analysis was performed to determine if regulation also
occurred at the protein level.
Figure 4.3 (Panel A) displays a representative hK4
Western blot of whole cell protein extracted from LNCaP cells treated with DHT, DHT
plus T3, and T3 alone. Two distinct bands of approximately 38 and 40 kDa are clearly
visible in the DHT and DHT plus T3 lanes, while the lower band in the control and T3
alone lanes is very faint. As the C terminus antibody, which detects both full length and
variant 1, was used in these experiments it is likely that the lower band corresponds to
the variant protein while the upper band corresponds to the full length protein.
Densitometry of the upper band, using β-tubulin as a housekeeping protein (Panel B),
was used to correct for uneven protein loading between lanes in each experiment.
Although a qualitative assessment would suggest that hK4 is up-regulated by DHT and
DHT plus T3, and down-regulated by T3, the densitometry histogram of six Western
blots (duplicates from three separate experiments) (Panel C) indicates that no significant
change occurred for full length hK4. Although densitometry was not performed for the
variant protein, due to difficulty in discriminating it from the upper band, it appears that
the expression of this protein is stimulated by the presence of DHT.
Figure 4.4 shows a representative Western blot for PSA using the polyclonal Dako
antibody. The two predominant bands at ~33 kDa and ~22 kDa represent pro-PSA and
87
Chapter 4
Control
DHT
DHT+T3
T3
52.9
~40 kDa
A
hK4
~38 kDa
35.4
B
54 kDa
52.9
β-Tubulin
Densitom etry
C
Fold Increase
1.5
1
0.5
0
Ctrl
DHT
DHT/T3
T3
Figure 4.3 The expression of hK4 protein in response to DHT and T3 treatment
Panel A displays a representative hK4 Western blot (with the C terminus antibody) of
whole cell protein extracted from LNCaP cells treated with DHT, DHT plus T3, and T3
alone for 48 h. Densitometric analysis (Panel C) is shown in the histogram below the
Western blots for the 40 kDa hK4 (presumed full length protein). The densitometry
(Panel C) is compiled from pooled data of duplicate Western blots from three
independent growth factor treated LNCaP whole cell lysate preparations. The signal for
hK4 was normalised for protein loading by the signal for the housekeeping protein, βtubulin (Panel B) on the same membrane. Protein marker sizes are indicated to the left of
Panels A and B.
88
Chapter 4
Control
A
DHT
DHT+T3
T3
34.7
~33 kDa PSA
~22 kDa
22
B
54 kDa
β-Tubulin
52.9
C
Densitometry
Fold Increase
2.5
2
1.5
1
0.5
0
Ctrl
DHT
DHT/T3
T3
Figure 4.4 The regulation of PSA protein by DHT and T3
Western blot analysis for PSA expression (Panel A) in whole cell lysates extracted from
LNCaP cells treated with DHT, DHT plus T3 or T3 alone. Densitometric analysis (Panel
C) is shown in the histogram below the Western blots for the ~33 kDa PSA (pro-form).
These data represent pooled data compiled from duplicate Western blots from three
independent experiments. The signal for PSA was normalised for protein loading by the
signal for the housekeeping protein, β-tubulin (Panel B) on the same membrane. Protein
marker sizes are indicated to the left of Panels A and B.
89
Chapter 4
an internally clipped PSA, respectively.
In addition, several other bands can be
observed and are considered artifacts due to non-specific antibody binding. Qualitative
assessment of the Western blots suggests that PSA is up-regulated by DHT and DHT
plus T3 in the same manner as secreted PSA protein (Figure 4.1) and PSA mRNA
(Figure 4.2, Panel A). However, densitometry analysis of the Westerns from the three
separate cell culture experiments indicates that no significant change occurred, despite
the apparent increases observed in the DHT and DHT plus T3 lanes.
4.2.2
Regulation of PSA and KLK4 in LNCaP cells by EGF
To assess the effect of EGF on PSA and KLK4 mRNA and protein levels, three
individual cell culture experiments were performed. Gene and protein expression was
determined by immunoassay (for PSA), quantitative RT-PCR and Western blot.
4.2.2.1 PSA assay of conditioned medium
The medium of LNCaP cells treated with increasing concentrations of EGF (0, 10, 50
and 100 ng/ml) was assayed for PSA protein levels by Greg Ward at the Princess
Alexandra Hospital, Brisbane.
Figure 4.5 displays the combined results of 3
independent experiments. Although a slight increase in secreted PSA was observed in
response to 10 ng/ml EGF treatment, no statistical significance was reached.
In
addition, the 50 and 100 ng/ml EGF treatments resulted in slightly less secreted PSA
than the untreated control. The results of the immunoassay indicate that PSA secretion
is not significantly regulated by EGF at the concentrations tested in these experiments.
This result is not consistent with the only previous report in which 50 ng/ml EGF
resulted in a significant decrease in PSA secretion from LNCaP cells (Henttu and Vihko,
1993).
4.2.2.2 PSA and KLK4 mRNA regulation by EGF
Quantitative PCR was performed on cDNA reverse transcribed from total RNA
extracted from LNCaP cells treated with increasing concentrations of EGF. In contrast
to a previous report (Henttu and Vihko, 1993), PSA transcripts increased significantly
(2.3 fold) in response to 50 ng/ml of EGF treatment (Figure 4.6A). Marginal, non
90
Chapter 4
PSA Secretion
1.4
Fold Change
1.2
1
0.8
0.6
0.4
0.2
0
0ng/ml EGF
10ng/ml EGF
50ng/ml EGF
100ng/ml EGF
Figure 4.5 PSA protein secretion in response to EGF treatment
A PSA immunoassay was performed on conditioned medium from three independent
experiments of LNCaP cells treated with increasing concentrations of EGF. Data are
presented as the fold increase over the untreated control (0ng/ml EGF) LNCaP cells
(assigned a value of 1). Standard error of the mean is indicated by bars and statistical
significance was assessed using a one-way ANOVA followed by Tukey Post-Hoc
analysis. However statistical significance between treatments was not reached.
91
Chapter 4
PSA mRNA
A
Fold Increase
4
*
3
2
1
0
0ng/ml
10ng/ml
50ng/ml
100ng/ml
EGF Concentration
B
KLK4 mRNA
4
*
**
10ng/ml
50ng/ml
3
2
1
0
0ng/ml
100ng/ml
EGF Concentration
Figure 4.6 The regulation of PSA and KLK4 mRNA by EGF
Quantitative PCR analysis of EGF treated LNCaP cells assayed for PSA (Panel A) and
“total” KLK4 (Panel B). Data are presented as the fold increase over 48 h untreated
control LNCaP cells (assigned a value of 1), with standard error of the mean indicated
by error bars. Quantitative PCR was performed in triplicate in three separate PCR runs
for three separate experiments for each growth factor treatment. Statistical significance
was assessed using a one-way ANOVA followed by Tukey Post-Hoc analysis (*P<0.01;
**P<0.001).
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Chapter 4
significant increases in PSA mRNA were also observed for the 10 and 100 ng/ml EGF
treatments. KLK4 mRNA displayed a similar up-regulation, however significant
increases in “total” KLK4 transcript were observed at 10 ng/ml EGF (2.6 fold) and 50
ng/ml EGF (2.9 fold) (Figure 4.6B). A small decrease in KLK4 mRNA in comparison to
the untreated control was observed in response to the 100 ng/ml EGF treatment.
4.2.2.3 PSA and hK4 protein expression in response to EGF treatment
Since both PSA and KLK4 were shown to be regulated by EGF at the mRNA level,
Western blot analysis was performed to determine if regulation also occurred at the
protein level. Figure 4.7 (Panel A) displays a representative hK4 Western blot of whole
cell protein extracted from LNCaP cells treated with increasing concentrations of EGF.
In all lanes there are two distinct bands of approximately 38 and 40 kDa. As with the
DHT/T3 experiment described above, the C terminus antibody, which detects both full
length and variant 1 hK4, was used in these experiments also. Densitometry of the
upper band (which likely corresponds to the full length protein) was normalised using βtubulin as a housekeeping protein, to correct for uneven protein loading between lanes in
each experiment.
Although no statistically significant changes were observed, the
histogram of the densitometric analysis of all three experiments reveals a similar pattern
of hK4 protein regulation to that of the KLK4 mRNA transcript regulation, with both 10
and 50 ng/ml EGF treatment resulting in an increase in hK4 compared to the untreated
control and decreased levels with 100 ng/ml EGF.
Western blot analysis for PSA was also carried out on the same membranes as used in
the hK4 analysis. Although two different PSA antibodies were used (Dako and Santa
Cruz) at varying concentrations on 6 separate blots, no signal was detected (data not
shown). Therefore, intracellular PSA protein could not be visualised or quantified for
changes in response to EGF treatment. It was thought that stripping the antibodies from
the membrane after hK4 and β-tubulin had been detected may have resulted in the
loaded protein also being removed from the membrane.
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0
A
10
50
100 ng/ml EGF
52.9
~40 kDa
hK4
~38 kDa
35.4
B
54 kDa
52.9
β-Tubulin
Densitometry
C
F o ld In crease
2
1.5
1
0.5
0
0 ng/ml EGF 10 ng/ml EGF 50 ng/ml EGF 100 ng/ml EGF
Figure 4.7 The expression of hK4 protein in response to EGF treatment
A representative Western blot analysis using the C terminus antibody for hK4
expression (Panel A) in whole cell lysates extracted from LNCaP cells treated with
increasing concentrations of EGF. Densitometric analysis (Panel C) is shown in the
histograms for the 40 kDa hK4 (full length). The histograms represent pooled data for
duplicate Western blots from three independent EGF treated LNCaP whole cell lysate
preparations. The signal for hK4 was normalised for protein loading by the signal for the
housekeeping protein, β-tubulin (Panel B) on the same membrane. Protein marker sizes
are indicated to the left of Panels A and B.
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4.3 DISCUSSION
Prostate cancer is rapidly becoming the most commonly diagnosed malignancy in men
in developed countries, and while androgen ablation therapy is the most effective
treatment for non-localised disease, it offers only temporary control. While the role of
androgen is important, alone it is insufficient to maintain cellular growth.
In the
prostate, complex interactions exist between androgens and many other growth factors
and hormones to support the growth of a developing tumour.
The expression,
regulation, and production of many of these growth factors and hormones are modified
in prostate cancer. Additionally, these hormones/growth factors are known to regulate
the expression levels of many genes important in prostate cancer. This study has
demonstrated the effects of DHT, T3 and EGF on the expression of “total” KLK4, which
was shown to be more highly expressed in malignant compared to benign prostate
tissues (Chapter 3).
Prior to the outset of these studies, few reports had detailed the expression of KLK4 in
response to hormones and growth factors in the LNCaP cell line (Nelson et al., 1999).
The androgen-responsive LNCaP cell line has been used extensively to study growth
characteristics of prostate cancer cells and gene regulation by a variety of growth
factors. Nelson et al (1999) used the LNCaP line as a model to evaluate KLK4
expression under androgenic control. At the Northern level, they found that 1 nM of
synthetic androgen, R1881, produced a 1.7 fold increase in expression in LNCaP cells
after 48 h. In the data presented here, the highly sensitive quantitative RT-PCR assay
was used to assess the regulation of KLK4 in the LNCaP model using the
physiologically active androgen, DHT. In this study, 10 nM DHT produced a 2.6 fold
increase in KLK4 transcript levels in comparison to the untreated control after 48 h
stimulation. The result of this study confirms the previous report that KLK4 is positively
regulated by androgen (Nelson et al., 1999). The biological responsiveness of the
experiment (ie that the LNCaP cells were appropriately stimulated by DHT) was
confirmed by demonstrating the expected rise in PSA protein secretion (Lee et al, 1995).
These results were further validated by demonstrating the expected increase in PSA
mRNA in LNCaP cells stimulated by DHT (Montgomery et al, 1992; Lee et al, 1995).
95
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In addition to the mRNA assessments, protein analysis of the whole cell extracts was
also performed. Both PSA and hK4 appeared to increase with DHT treatment, however
densitometric analysis revealed no statistically significant change. While mRNA levels
do not always parallel protein levels (De Moor and Richter, 1999), it is also possible that
once PSA and hK4 are translated, they are secreted from the cell soon after, accounting
for the low levels of protein intracellularly. Considering the high levels of PSA protein
secretion observed, as determined by the PSA immunoassay, this is a likely possibility.
Currently an hK4 immunoassay is not available, hence the levels of hK4 secreted into
the media were not able to be assessed in the same manner. However, it would be useful
to examine the secreted hK4 levels via Western analysis. Another explanation for the
discrepancy between the mRNA and protein data is that only the upper band which is
thought to correspond to the full length hK4 protein was analysed by densitometry,
while the quantitative RT-PCR results take into account full length KLK4 and all mRNA
variants. It is possible that the androgen regulation observed may be due to one or more
of the variants, in particular, the exon 1 deleted form which is abundantly expressed in
the LNCaP cell line.
Since the earlier report (Nelson et al., 1999), further regulation studies have been
performed by other groups to assess the expression levels of KLK4 in response to
androgen treatment. Treatment of LNCaP cells with 10 nM R1881 for 24 h resulted in
an approximate 18 fold increase in KLK4 mRNA as determined by Northern blot
analysis (Korkmaz et al., 2001). The higher fold change of KLK4 mRNA observed by
the Korkmaz study (2001) compared to both this study and the Nelson study (1999) may
reflect the time course of the experiment, as the Korkmaz study was conducted over 24
h, while this study and the Nelson study was conducted over 48 h. As Korkmaz et al
(1999) found a greater increase in mRNA in a shorter time period, this may suggest that
the regulation of KLK4 may be at the transcriptional level, like PSA and KLK2, through
the binding of androgens to an androgen response element (ARE) (Riegman et al, 1991;
Young et al, 1995; Cleutjens et al, 1997).
96
However, in order to confirm this, it is
Chapter 4
necessary to perform time course experiments ranging from 2 - 24 h which may indicate
whether regulation is occurring at the transcriptional level or not.
In addition to the reported up-regulation of KLK4 in the LNCaP model, in this study and
others (Nelson et al., 1999, Korkmaz et al., 2001), up-regulation of KLK4 has been
demonstrated in the breast carcinoma cell line, BT-474, by DHT as well as estradiol
(Yousef et al, 1999). Therefore, it is clear that KLK4 is regulated by androgens in both
the prostate and the breast.
Many studies, in vitro and in vivo, have related thyroid hormones and human cancer
since the use of thyroid extracts for breast cancer treatment was described more than a
century ago (Beatson, 1896). The data indicate that thyroid status and disease affect
tumour formation, growth and metastasis in experimental animals and humans (Lemaire
and Baugnet-Mahieu, 1986, Smyth, 2003), although little research has focused on the
effects of thyroid hormone with respect to prostate cancer. Previous reports indicating
that thyroid hormone receptor is expressed in prostate tissues and cell lines (Sakurai et
al., 1989, Esquenet et al., 1995) coupled with the suggestion that T3 is critical for
supporting the growth of prostate cancer cells in vitro (Hedlund and Miller, 1994)
suggests an important role for thyroid hormones in prostate carcinogenesis. In this
study, the possible interactive effect of androgen with triiodothyronine on KLK4 and
PSA expression was also assessed.
As discussed above, PSA secretion in response to androgen increased significantly over
the control. However a greater response was observed in response to androgen together
with T3. Alone, T3 had little effect on PSA secretion. Although the PSA secreted in
response to the combined treatment of DHT with T3 was not statistically significant, the
additional increase observed over the DHT alone treatment parallels previous reports
where T3 in the presence of androgen enhanced the androgen-induced up-regulation of
PSA (4 fold increase), while T3 alone produced only a marginal increase (less than 2
fold)(Zhang et al., 1999, Zhu and Young, 2001). While a 20 fold increase in PSA
secretion was observed in this study upon DHT and T3 treatment, a number of
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Chapter 4
differences in the experimental procedures exist between this study and that of Zhang
(1999). This study demonstrated the effects of androgens with 10 nM DHT, while the
Zhang study used 1 nM Mibolerone. Additionally, the spent medium was collected after
48 h of hormone/growth factor treatment in this study, whilst in the Zhang study, the
spent medium was collected 7 days post stimulation, which may have resulted in
considerable PSA protein degradation during that time.
Once it was established that the cells were responsive, in accordance with the literature,
the transcript levels of PSA and KLK4 were assessed by quantitative RT-PCR. In this
study, both PSA and KLK4 were found to be positively regulated by the combination of
DHT and T3.
Additionally, when compared with the control value, the increase
observed for KLK4 mRNA was more significant for DHT in combination with T3 than
for T3 alone. In response to T3 only, no significant changes in transcript levels were
detected for either gene. These data parallel that of the earlier study where it was
demonstrated that T3 further increased the androgenic induction of PSA, but not that of
KLK2, and that T3 alone had no significant effect on transcriptional activity of either
PSA or KLK2 (Zhang et al., 1999).
Taken together, the above results suggest PSA and KLK4 are regulated in a similar way
by DHT and DHT in combination with T3, while KLK2 does not respond in the same
manner to these hormones/growth factors.
Considering that T3 alone produces no
increase in PSA or KLK4 transcript levels, yet in combination with androgen a greater
increase is observed than with androgen alone, it was thought that the mechanism by
which this occurs may be via T3 increasing androgen receptor levels in the cells.
However it has since been proven that T3 does not increase the level of androgen
receptor in the LNCaP cell model (Zhang et al., 1999). Clearly, further studies are
required to elucidate the mechanism by which T3 potentially enhances androgen
induction of PSA and KLK4.
The intracellular protein levels, as evidenced by Western blot, appeared to parallel the
quantitative RT-PCR data, although the densitometry analysis revealed the changes to be
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non-significant. As mentioned above, it would be useful to assess the secreted levels of
hK4 in the media to determine whether the low intracellular levels are due to the
protein’s rapid secretion from the cell.
In a recent study, expression of the exon 1 deleted KLK4 transcript was assessed in
response to multiple hormones (Korkmaz et al, 2001). The exon 1 deleted variant was
found to be upregulated by androgen, estrogens, progestins and dexamethasone but not
by vitamin D3 or thyroid hormone in the LNCaP cell line after 24 h of hormone
stimulation (Korkmaz et al, 2001). Considering that the results of this chapter assessed
“total” KLK4, and therefore include the exon 1 deleted transcript, the findings of the
Korkmaz study with respect to the androgen and T3 treatments also correlate with the
findings of this chapter. It would be useful to assess each transcript in isolation in order
to determine the hormone/growth factor responsiveness of each KLK4 variant. However
this may prove difficult considering it is not possible to design primers to detect each
variant separately.
EGF is found at elevated levels in high-grade PCa tumours (Harper et al., 1993; GlynneJones et. al., 1996). Furthermore, EGF is necessary for human prostate epithelial cells to
survive in serum-free medium in primary culture (Peehl et al, 1989) and increased
expression of EGF has been linked to prostate cancer development (Fowler et al, 1988).
One pathway by which EGF has been linked to the development of this disease is
through the matrix metalloproteases. Interestingly, EGF has been shown to increase
mRNA and secreted protein levels of MMP-7 (matrilysin) in the LNCaP cell line
(Sundareshan et al., 1999). Matrilysin has been suggested to be involved in invasion
and metastasis of prostate cancer due to the ability of matrilysin cDNA-transfected
prostate cancer cell lines to invade the diaphragm in a severe combined immunodeficient
mouse model (Powell et al., 1993). In this study, PSA and KLK4 expression levels were
assessed in response to increasing concentrations of EGF.
While no appreciable changes were observed in the PSA secretion data, when PSA
mRNA was assessed, a significant increase in transcript levels occurred in response to
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Chapter 4
EGF stimulation (50 ng/ml). However this result differs from that of a previous study
which showed that EGF caused a significant decrease in PSA transcript levels at the
same concentration (Henttu et al, 1993). For KLK4 mRNA, significant increases in
transcript levels were observed but the increases were apparent with lower EGF
concentrations (10 and 50 ng/ml) than that seen with PSA. When the intracellular
protein levels were assessed, the same pattern was seen on the Western blots, although
these were not significant changes when analysed by densitometry. Due to technical
difficulties, PSA was unable to be detected on the Western blots. One previous study
which examined the effect of EGF on PSA found that PSA levels decreased in response
to EGF treatment (Henttu et al, 1993), which indicates that this may be the cause of the
difficulty in detecting the protein.
Nonetheless, this study has demonstrated that KLK4 mRNA levels are positively
regulated by EGF.
Since EGF expression is known to be regulated by androgen
(Hiramatsu et al, 1988, Nishi et al, 1996), it would be interesting to determine the
interactive effects of androgen in combination with EGF on KLK4 mRNA and protein
expression. Additionally, it would also be of interest to examine the effect of TGFβ1 in
combination with EGF on KLK4/hK4 expression levels. While it is well established that
EGF stimulates the proliferation of LNCaP cells (Schuurmans et al., 1988, Connolly and
Rose, 1990, MacDonald and Habib, 1992, Henttu and Vihko, 1993) the effect of TGFβ1
on the growth of LNCaP cells is controversial. TGFβ1 by itself has no growth effects on
LNCaP cells (Schuurmans et al., 1988, Wilding et al., 1989), but has been observed to
block the induction of growth of LNCaP cells by EGF (Schuurmans et al., 1988, Henttu
and Vihko, 1993), and to mediate androgen-regulated growth arrest in LNCaP cells
(Kim et al., 1996).
In summary, this chapter has demonstrated the regulation of KLK4 expression by the
hormones/growth factors, DHT, DHT in combination with T3 and EGF. As previous
studies have demonstrated that KLK4 expression is also increased in response to
progestins, estrogens and dexamethasone in the androgen responsive LNCaP cell line,
the information presented here adds to the earlier data indicating that multiple hormonal
100
Chapter 4
and growth factor signals can influence KLK4 expression in the prostate. Clearly,
hormonal and growth factor regulation of genes involved in prostate cancer is a complex
matter and further studies are required in order to fully elucidate the significance of the
regulation observed. While all these studies were carried out in the androgen sensitive
LNCaP cell line, it would be interesting to examine the effect of these and other growth
factors/hormones in other cell lines, such as DU145 and PC-3 which are representative
of androgen insensitive disease.
101
CHAPTER FIVE
THE ESTABLISHMENT OF STABLY TRANSFECTED
PC-3 PROSTATE CANCER CELLS
OVER-EXPRESSING FULL LENGTH KLK4
Chapter 5
5.0 INTRODUCTION
Previous chapters in this thesis have indicated that the recently identified KLK4 gene is
expressed in a number of prostate cell lines and is more highly expressed in prostate
cancer tissues than benign prostatic tissue. Furthermore, it is regulated by several key
hormones/growth factors (DHT, DHT plus T3, EGF) that are known to be important in
the maintenance and development of both the normal and malignant prostate. However,
what remains to be established is the biological function of KLK4/hK4 in prostate cancer
cells.
Previous studies have proposed various enzymatic actions of hK4, including its ability to
activate pro-PSA and single chain urokinase-type plasminogen activator (scuPA, prouPA) (Takayama et al., 2001b). Additionally, it was shown to completely degrade the
seminal plasma protein, prostatic acid phosphatase, but failed to cleave serum albumin,
another protein from human seminal plasma (Takayama et al., 2001). The authors
suggested that hK4 may have a role in the physiological processing of seminal plasma
proteins such as pro-PSA and PAP, as well as in the pathogenesis of prostate cancer
through its activation of pro-uPA. The data described were derived from biochemical
studies with recombinant hK4 and imply that hK4 may be involved in various functional
aspects of cancer progression, namely invasion, via degradation of the ECM through
uPA activation, but as yet this has not been confirmed using in vitro functional assays.
Therefore, to determine other potential roles of hK4 in the process of tumourigenesis, it
was necessary to develop a cellular model in order to assess tumourigenic parameters. A
classic approach to determine the function of newly identified genes is to establish either
over-expressing or under-expressing in vitro and/or in vivo models.
In vitro models of prostate cancer are a vital resource due to their flexibility in culture
and their ability to be transfected with a gene of interest (Mitchell et al., 2000). This
chapter describes the development of an in vitro, over-expressing cell model via the
stable transfection of the PC-3 cell line with a full length KLK4 expression construct.
PC-3 cells were chosen as they do not express full length KLK4 (see Chapter 3).
Although this cell line expresses three KLK4 transcripts, this was not considered to pose
103
Chapter 5
a major problem as qualitative analysis of the mRNA transcripts and proteins suggests
they are expressed at low levels in this cell line compared with other prostate cancer cell
lines (Chapter 3).
As the PC-3 cell line was originally derived from a lumbar vertebral bony metastasis and
classified as a poorly differentiated prostatic adenocarcinoma (Kaighn et al., 1979),
these cells represent a good model to study the effect of hK4 in aggressive prostate
cancer. It is well known that PC-3 cells are the most invasive original line (compared
with the LNCaP and DU145 cell lines), and are tumourigenic when injected into
immunosuppressed mice (Kaighn et al., 1979, Shevrin et al., 1988, Rembrink et al.,
1997). Many other genes have been over-expressed in this line, including the androgen
receptor (Dai et al., 1996, Heisler et al., 1997, Snoek et al., 1998, Shen et al., 2000), cell
adhesion molecules such as C-CAM (Hsieh et al., 1995) and α-catenin (Ewing et al.,
1995), and prostate-specific molecules such as PSA (Balbay et al., 1999), prostatic acid
phosphatase (Lin et al., 1998) and prostasin (Chen et al., 2001). Therefore, this cell line
was chosen as a suitable model in which to over-express full length KLK4 in order to
subsequently assess the functional significance of KLK4/hK4 over-expression.
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Chapter 5
5.1 MATERIALS AND METHODS
5.1.1 KLK4 construct and mammalian expression vectors
A full length pre-pro-KLK4 construct in the pcDNA3.1/V5-His (Invitrogen) mammalian
expression vector, previously prepared by Dr Tracey Harvey, was used in this study to
create stable KLK4 over-expressing PC-3 clones. The KLK4 construct consists of the
full length KLK4 cDNA, which, when translated into protein, yields the pre-pro-enzyme.
The vector pcDNA3.1/V5-His incorporates a 14 amino acid V5 epitope and a 6 amino
acid Histidine tag at the C terminus. Figure 5.1 illustrates a schematic diagram of the
KLK4 construct inserted in pcDNA3.1/V5-His and also the vector only control which
was prepared without any cDNA inserted into the vector. Figure 5.1 also shows the
amino acid sequence for the hK4 protein which highlights the pre, pro and mature
protein regions and the V5 and His tags.
5.1.2 Lipid-mediated transfection
The general details of PC-3 cell culture are summarised in the Materials and Methods
chapter, Section 2.2.1; however, precise details pertaining to specific experimental
procedures are outlined below.
Selection antibiotics allow stably transfected cell lines to be created as they select cells
that contain the transfected vectors which express resistance genes. Cells expressing
pcDNA3.1 (V5/His) constructs containing the neomycin resistance gene required the
selection antibiotic, geneticin.
To determine PC-3 cell sensitivity to geneticin, a dose response study was performed.
This involved incubating PC-3 cells in increasing concentrations of geneticin in medium
containing 10% FBS, changing the medium every 3 days and observing the percentage
of cells which did not survive.
A concentration of 100 µg/ml geneticin killed
approximately 50% of cells after a period of 2 weeks (data not shown).
105
Chapter 5
A
BamHI – KLK4 - XhoI
Vector only
control
KLK4
construct
B
MATAGNPWGWFLGYLILGVAGSLVSGSCSQIINGEDCSPHSQPWQAALVMENELFCSGVLVHPQW
VLSAAHCFQNSYTIGLGLHSLEADQEPGSQMVEASLSVRHPEYNRPLLANDLMLIKLDESVSESD
TIRSISIASQCPTAGNSCLVSGWGLLANGRMPTVLQCVNVSVVSEEVCSKLYDPLYHPSMFCAGG
GQDQKDSCNGDSGGPLICNGYLQGLVSFGKAPCGQVGVPGVYTNLCKFTEWIEKTVQASVSSLEG
PRFEGKPIPNPLLGLDSTRGHHHHHH
Figure 5.1 Vector schematics and complete amino acid sequence of hK4 expression
construct
A. Schematic diagrams of the vector only control and the KLK4 construct when inserted
into pcDNA3.1 (V5/His) indicating the restriction enzyme sites in the multiple cloning
site.
B.
Amino acid sequence of the hK4 expression construct.
The pre-peptide is
underlined, the pro-peptide is double underlined and the mature protein is italicised. All
pcDNA3.1 vector residues are in bold font with the V5 epitope in bold italics and the
His tag in underlined bold.
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Chapter 5
Figure 5.2 illustrates a schematic representation of the transfection procedure and culture
conditions. Twenty-four hours prior to transfection, the PC-3 prostate cancer cell line
was passaged into T25 cm2 flasks in medium and allowed to grow up to 95%
confluency. A high confluency is required because approximately one cell out of 1x104
cells will incorporate the transfected plasmid DNA (pDNA) into the chromosomal DNA.
The monolayer was washed 3 times in sterile PBS to remove residual antibiotics
(penicillin/streptomycin) and 2 ml of antibiotic free RPMI medium was added to the
cells. Meanwhile, 0.5 ml antibiotic free and serum free medium was incubated with 60
µl Lipofectamine 2000 Reagent (LF2000; Invitrogen) for 5 min. Four micrograms of
pDNA (KLK4 in pcDNA3.1, or the vector only control) was then added and the
incubation continued for 20 min to allow the DNA to bind to the LF2000.
The
complex was then added directly to the cells and incubated overnight in a humidified
37ºC incubator.
5.1.3 Generation of stably transfected clones
Following the overnight incubation in LF2000, transfected cells were washed in PBS
and trypsinised to detach them from the flask. Cells were resuspended in medium
containing antibiotics and 10% FBS, and split into a 24-well plate where they were
incubated for a further 24 h at 37ºC. The next day, the medium was removed and
replaced with medium containing 100 µg/ml geneticin to begin selection of transfected
cells. The antibiotic-containing medium was replaced every 3 days, and after 2-3 weeks
single colonies of cells were apparent. When several colonies from different wells were
of a reasonable size (~100 cells), they were washed in PBS, trypsinised and counted, and
the cells were divided into the desired split of a theoretical concentration of 0.3
cells/well. This concentration of cells therefore allowed only single cells to be grown in
96-well plates. In the case that more than one cell was seen growing in a single well, the
well was eliminated and not allowed to grow further. Once the single cells grew to
100% confluency, they were passaged directly into a 24-well plate and grown to
confluency once more before being passaged into a 6-well plate. Finally, the cells were
moved into a T25 cm2 flask and progressively split until there were enough cells for
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Chapter 5
KLK4 DNA
Diluted LF2000
LF2000 and DNA combined
Incubate 20 min at RT
Add to cells in antibiotic-free medium
containing 10% FBS
Incubate for 24 hours
Passage into 24 well plates and after 24
hours incubate in growth medium
containing selection antibiotics
Select colonies and passage into 96-well plates at a theoretical
split of 0.3 cell/well for single cell colonies to form
. . .
.
When confluent, culture in presence of selection antibiotics,
progressively passage into large area plates and flasks for
experimentation and preserving
96 well
plate
24-well
plate
6-well
plate
Figure 5.2 Lipid-mediated transfection protocol
Key: LF2000 – Lipofectamine 2000
108
T25cm2 flask
Chapter 5
cryo-preservation (Materials and Methods, Section 2.2.1.3). After 3 months, 5 vector
only clones and 6 KLK4 clones were still viable and preserved.
5.1.4 Confirmation of stably transfected clones
To establish that the clones stably transfected with KLK4 constitutively expressed the
mRNA and protein, conventional and quantitative RT-PCR, Western blot analysis and
immunofluorescence were performed.
5.1.4.1 Collection of cell pellets and conditioned media
To collect transfected cells for RNA and protein extraction, cells were grown to ~7080% confluency in T80 cm2 flasks. Cells were first washed in PBS, and then 5 ml of
serum-free medium was added, before incubation for 48 h in a humidified 37ºC
incubator. After this period, the conditioned medium was removed and transferred to a
clean tube, whilst the cell monolayer was trypsinised and resuspended in serum-free
medium and centrifuged at 1000 rpm for 5 min. The supernatant was removed by
aspiration and the pellet was stored at -70ºC until needed. Conditioned medium was
centrifuged to remove cell debris at 1000 rpm for 5 min, divided into 1 ml aliquots, and
stored at -20ºC.
5.1.4.2 RT-PCR
Total RNA was extracted from 48 h serum-starved cell pellets and reverse transcription
was performed on 5 µg of RNA from each clone (Section 2.2.2 and 2.2.3). To confirm
the over-expression of the transfected full length KLK4 transcript, PCR primers
producing a product spanning exons 1 -5 were used and are outlined in Table 3.2. These
primers amplify only full length KLK4 and therefore would not detect the exon 1 deleted
KLK4 transcripts. Primers were also used for the amplification of β2-microglobulin to
detect for genomic contamination in the cDNA sample, and a negative control (no
cDNA) was used in every PCR to rule out reagent contamination.
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Chapter 5
5.1.4.3 Quantitative RT-PCR
Real-time PCR was carried out on the Idaho Technology ‘Light Cycler’ LC32 (Idaho
Technology, Inc., Utah, USA) as described in Section 2.1.3.2. Primer combinations and
cycling conditions for KLK4 and β2-microglobulin are indicated in Table 3.3. To detect
full length KLK4, primers spanning exons 1-3 were used producing a 250 bp product
which is within the range of acceptable product sizes for quantitative analysis on the
LC32 Light Cycler.
Each assay was completed twice, in quadruplicate, and copy
numbers were normalised to β2-microglobulin levels, collected and averaged for each
individual clone by dividing the gene copy number (ie. KLK4) with the β2-microglobulin
copy number gained for each individual clone.
Normalised ratios for each clone
population were averaged and statistical analyses were performed using a two-tailed
Student’s t-test.
5.1.4.4 Western blotting
Western blotting was initially performed to determine whether the inserted cDNA
transfected into the cells was translated into protein and secreted. Both cell lysates and
conditioned medium from hK4 over-expressing clones were examined.
Whole cell lysate was extracted from 48 h serum-starved cell pellets and 10 µg of the
extracted protein was mixed with 2X loading buffer, and loaded into the wells of a 10%
SDS-PAGE gel, as described in Chapter 2, Sections 2.2.6.1 and 2.2.6.2.
To determine whether the cells secreted hK4, spent medium from selected clones was
initially concentrated, as per Chapter 2, Section 2.2.6.1. Ten microlitres of concentrated
medium with 2X loading buffer was loaded and electrophoresed on a 10% SDS-PAGE
gel. The Western blots were incubated overnight at 4ºC with the N terminus hK4
antibody (which detects full length hK4). This antibody also detects variants 3 and 4,
although these are present in very small quantities in PC-3 cells, as determined by
immunofluoresence (see Chapter 3). Western blotting was then completed as described
in the Materials and Methods, Section 2.2.6.3.
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Chapter 5
5.1.4.5 Immunofluorescence
Immunofluorescence was carried out to further confirm the over-expression of full
length hK4 and to establish whether the stably transfected clones expressed hK4 equally
throughout the monolayer. This involved growing each clone in 8-well or 16-well
Labtek chamber slides (Medos) in medium containing 10% FBS. The precise details for
this method are listed in the Materials and Methods, Section 2.2.7. In addition to the N
terminus antibody which detects full length hK4, the C terminus antibody was also used
(both at 1:1000 dilution). This antibody recognises both the full length and the N
terminally truncated variant 1 protein and was used to qualitatively assess the staining
patterns between the transfected and non-transfected cells with respect to the full length
protein and the variant 1 form.
5.1.5 Morphological analysis
Phase-contrast photomicrographs were taken for hK4 over-expressing clones to observe
any alterations in cell morphology and phenotype when compared to the PC-3 native
cells and vector only controls. Cells were grown in T25 cm2 or T80 cm2 flasks to 70100% confluency in medium containing 10% FBS and photographs were taken using a
Leitz TMS-F inverted microscope (Leica Microsystems) with a Nikon MPS30
microscope camera (Coherent Life Sciences).
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Chapter 5
5.2
RESULTS
5.2.1 Generation of stably transfected clones
Several stably transfected clones were generated - five vector only clones (V1, V2, V3,
V4, V5) and six KLK4 over-expressing clones (K4#2, K4#7, K4#8, K4#9, K4#10,
K4#11). For subsequent experiments, only two vector only clones were analysed (V1
and V3). At the outset, up to 24 individual clones were taken for each, but several
clones ceased proliferation while passaging through plates of increasing size (eg. 96 well
plate → 24 well plate; as described in Section 5.2.3). These clones were discarded
leaving the remaining viable clones to be cultured, preserved and used in future
experiments.
5.2.2 RT-PCR expression of KLK4 in transfected clones
To confirm that the transfected construct was successfully integrated into the PC-3 cell
line, RT-PCR was performed for 35 cycles on RNA extracted from the cells. To
distinguish endogenous KLK4 transcripts from transfected full length KLK4, primers
producing a product spanning exons 1-5 were used. Figure 5.3 (Panel A) displays the
ethidium bromide stained gel photograph which demonstrates that the clones transfected
with full length KLK4 are positive for this transcript (642 bp), while the native PC-3
cells and vector only controls were negative as they do not endogenously express the full
length transcript (see Chapter 3). The expression profile of β2-microglobulin showed
that all samples were of a similar quality, with no genomic contamination, giving an
expected size of 238 bp (Figure 5.3, Panel B).
5.2.3
Quantitative RT-PCR expression of KLK4 in transfected clones
Quantitative RT-PCR was performed to determine the transcript copy number of each
clone compared to the native PC-3 cells. Each raw value obtained from the real-time
PCR was normalised to the mean DNA copy number gained from assaying β2microglobulin quantitatively from each clone, vector control or non-transfected control
(native), and calculating a ratio between the two genes for each cell population. The
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Chapter 5
KLK4 Over-expressing Clones
Controls
A
B
M
–ve
N
V1
V3
2
7
8
9
10
11
872
603
642 bp
517
298
201
238 bp
Figure 5.3 RT-PCR analysis of full length KLK4 expression in transfected clones
Ethidium bromide stained gels illustrating mRNA expression of KLK4 (Panel A) and β2microglobulin (Panel B) in PC-3 stably transfected clones. Six KLK4 over-expressing
clones are shown, labeled 2, 7, 8, 9, 10 and 11. Vector only controls, V1 and V3, and
PC-3 native (N) were included as negative controls along with the no cDNA control
(-ve). Product sizes are indicated to the right of each gel. The marker is indicated to the
left. Key: M – Roche Marker IX for Panel A; Marker X for Panel B.
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Chapter 5
ratio for each KLK4 clone and vector control was then expressed in comparison to the
native cells.
Figure 5.4 illustrates that clone K4#8 expresses the greatest quantity of KLK4 mRNA
with a 172 fold increase over the native cells, which expressed negligible copies of full
length KLK4, as expected. Both K4#10 and K4#2 had a 57 fold increase followed by
K4#7 with a 49 fold increase, K4#11 with a 33 fold increase and finally K4#9 with a 6
fold increase in transcript copy number over the native cells.
All clones had a
statistically significant increase in transcript copy number over the native PC-3 cells.
Although the increase in the K4#9 clone was significantly greater than the native copy
number, it was also significantly less than the copy number of the other KLK4 clones.
Both vector only controls had slight increases in transcript copy numbers but were not
significantly different from the native cells as anticipated.
5.2.4 Protein analysis of hK4 in transfected clones
Western blot analysis was performed to confirm the over-expression of hK4 protein
within the cells. Whole cell lysate preparations were analysed using the N terminus antihK4 antibody (Figure 5.5, Panel A), which recognises full length hK4. One band of ~38
- 40 KDa can be seen in all of the clones with the greatest amount of protein in K4#7
and K4#8. The band size is slightly greater than the size for the porcine active hK4
enzyme which migrates as a doublet of 34 and 37 KDa on SDS-PAGE (Simmer et al.,
1998, Ryu et al., 2002), which suggests the band detected is pro-hK4. No protein was
detected in the native and vector only cells as expected. The housekeeping gene βtubulin was visualised on the same membrane to determine protein loading variability
between lanes (Panel B).
To determine whether hK4 was secreted from the clones, 20 x concentrated conditioned
medium was assessed using the N terminus antibody. Panel C displays the results which
show that one band of ~35 KDa was detected in all hK4 clones, with K4#8 secreting the
greatest amount of protein. Based on the reports of porcine K4 (Simmer et al., 1998,
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*
180
150
Ratio
120
90
*
60
*
*
30
*
*
KK4
4##
1111
KK4
4##
99
KK4
4##
1100
KK4
4##
88
KK4
4##
77
KK4
4##
22
VV3
3
-30
VV1
1
NNa
att
iivv
ee
0
Figure 5.4 Quantitative RT-PCR analysis of KLK4 expression in transfected PC-3
cells
Graphical representation of real-time PCR analysis of KLK4 mRNA in PC-3 native,
vector only and KLK4 over-expressing cells. Data are presented as the ratio of the
averaged KLK4 transcript copy number (with the native cells normalised to 1.0) divided
by the averaged β2-microglobulin transcript number with standard error of the mean
(bars).
Statistical significance was analysed using the Student’s t-test (* indicates
p<0.0001).
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Controls
N
52.9
A
V1
hK4 Over-expressing Clones
V3
2
7
8
9
10
11
38 - 40 KDa
35.4
92
B
54 KDa
52.9
hK4 Over-expressing Clones
Controls
N
C
V1
V3
7
8
35.4
9
10
11
35 KDa
Figure 5.5 Western blot analysis of K4 over-expressing clones and control cells
hK4 expression in the whole cell lysate from hK4 over-expressing PC-3 clones using the
N terminus hK4 antibody which detects full length hK4 (Panel A). One band can be
observed at ~38 KDa in all hK4 over-expressing cells. No protein is visible in the PC-3
native and vector only cells. A β-Tubulin blot illustrates protein loading variability
between sample wells (Panel B). Panel C displays the hK4 expression in the 20x
concentrated conditioned medium from the hK4 over-expressing PC-3 cells using the N
terminus hK4 antibody. One major band is seen at 35 KDa in all transfected clones to
varying degrees, while no protein can be observed in the PC-3 native and vector only
clones.
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Ryu et al., 2002), the size of the protein detected suggests that the active form of the
protein is secreted. Again, the native and vector only controls were negative.
5.2.5 Immunofluorescence
Figure 5.6 shows photomicrographs which further confirm the over-expression of hK4
protein in the cells stably transfected with the KLK4 construct. Immunofluorescent
staining detected using the N terminus antibody (Figure 5.6), which recognises full
length hK4 and variants 3 and 4, revealed the stably transfected clones exhibited the
greatest intensity of hK4 staining consistently throughout the monolayer in comparison
to the native and vector only cells. The staining is predominantly cytoplasmic, however
K4#2, K4#7 and K4#11 exhibited a possible perinuclear localisation. This suggests that
the increased staining is due to the transfected hK4, while the weak staining is attributed
to endogenous variants 3 and 4 as determined previously (see Chapter 3). Although not
a quantitative assessment, the greatest intensity of staining was observed in clones
K4#2, K4#7, K4#8, K4#10 and K4#11; whilst K4#9 had very little staining. As
expected, the native and vector only cells, which do not express full length KLK4/hK4
had negligible staining.
Using the C terminus antibody (Figure 5.7), which recognises both full length and
variant 1 hK4, immunofluorescent staining for hK4 in the PC-3 transfected cells was
compared to that in the PC-3 native and vector only cells. As expected, the cells
transfected with full length KLK4 exhibited more intense staining than the native and
vector only clones. K4#8, K4#10 and K4#11 had the greatest intensity of staining;
K4#2 and K4#7 had weaker staining, while K4#9 had very little staining. All staining
displayed a predominant cytoplasmic localisation. PC-3 native cells and the vector only
clones 1 and 3 showed some staining for hK4, indicative of the endogenous variant 1
hK4.
5.2.6 Cell morphology
Figure 5.8 illustrates the cell morphology of the PC-3 native cells, vector only control
clones and hK4 clones. In general, the cell morphology of the native, vector only
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PC-3 Native
Vector-only 1
Vector-only 3
K4#2
K4#7
K4#8
K4#9
K4#10
K4#11
Figure 5.6 Immunofluorescence analysis for hK4 expression in KLK4 transfected
PC-3 cells with the N terminus anti-hK4 peptide antibody
Immuno-staining using an N terminus anti-hK4 peptide antibody, which detects full
length hK4, is indicated by the green Alexa-Fluor stain. Immunofluorescent staining is
present in the hK4 over-expressing clones K4#2, K4#7, K4#8, K4#10 and K4#11. Little
staining is visible in the K4#9 cells and the PC-3 native and vector only controls.
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PC-3 Native
Vector-only 1
Vector-only 3
K4#2
K4#7
K4#8
K4#9
K4#10
K4#11
Figure 5.7 Immunofluorescence analysis for hK4 expression in KLK4 transfected
PC-3 cells with the C terminus anti-hK4 peptide antibody
Immuno-staining using a C terminus anti-hK4 peptide antibody, which detects both full
length and the N terminus truncated hK4, is indicated by the green Alexa-Fluor stain.
Immunofluorescent staining is present in the hK4 over-expressing clones K4#2, K4#7,
K4#8, K4#10 and K4#11. Less staining is visible in the PC-3 native, vector only
controls and K4#9 cells.
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PC-3 Native
Vector-only 1
Vector-only 3
K4#2
K4#7
K4#8
K4#9
K4#10
K4#11
Figure 5.8 Cellular morphology of KLK4 transfected PC-3 cells
PC-3 native cells, vector only control clones and K4#9 exhibit quite rounded cellular
morphology, while the remaining hK4 over-expressing clones (K4#2, K4#7, K4#8,
K4#10 and K4#11) have a spindle shape with many processes.
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control clones and K4#9 are similar in shape, with rounded cells and few processes. In
contrast, the remaining hK4 over-expressing clones appear to be more spindle shaped,
have numerous processes and are slightly larger in size than the parental PC-3 cells.
In addition to their morphological differences, the PC-3 native cells, vector only clones
and K4#9 displayed other phenotypic differences. These cells attached exceptionally
well to tissue culture flasks, were difficult to trypsinise and took greater than ten minutes
to release from the flask. They grew in confluent islands with significant cell-to-cell
contact, and on reaching 100% confluency, the PC-3 native, vector only and K4#9
clones formed a single even monolayer. In contrast, it was observed that the remaining
hK4 over-expressing clones had compromised adhesion to the culture surface on which
they were grown, and their release from the flask was generally instantaneous after the
application of trypsin. They also grew in a dispersed manner independently of adjacent
cells and demonstrated significantly less cell-to-cell contact. When approaching 100%
confluency, the hK4 over-expressing PC-3 clones had regions of available growth
surface between adjacent cells that remained exposed.
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5.3
DISCUSSION
This Chapter describes the stable transfection of hK4 into PC-3 cells. Initial
characterisation of these cells indicates that the introduced KLK4 cDNA is integrated
into the genome and the cells produce the hK4 protein to various levels as determined by
conventional and quantitative RT-PCR, Western blotting and immunofluorescence.
Stably transfected cell lines are a valuable tool in the study of prostate cancer when
investigating cellular functions including proliferation, adhesion, invasion and
migration.
Transfected expression constructs are usually incorporated into the
chromosomal DNA or maintained as an episome, and the amount of construct integrated
depends on the efficiency of transfection and the cell type used (Ausubel et al., 1994).
When examining the effects of over-expressed genes on cellular functions, it is
important to note that clonal variation limits the interpretation of the results obtained,
due to the differences in construct incorporation into the genome and initial
characteristics of the cell. In order to overcome this, several clones are examined when
assessing functional profiles of stably transfected cells.
Therefore, six hK4 over-
expressing clones were created, characterised and later utilised in functional analyses
(see Chapter 6).
RT-PCR analysis of the KLK4 over-expressing clones demonstrates that each clone
expresses full length KLK4, at varying levels. The PC-3 native cells and vector only
clones do not express the full length KLK4 transcript (see Chapter 3). In order to assess
the transcript levels, quantitative RT-PCR was carried out. The results show that all
KLK4 over-expressing clones are expressing significantly greater number of transcripts
than the control clones. Interestingly a vast difference exists in the transcript levels
between clones; namely, K4#8 and K4#9, which express the highest and lowest
transcript levels respectively. The difference observed is most likely due to transfection
efficiency. Nevertheless, the six KLK4 clones produced represent a good range of
clones to assess cellular function as four of the clones have approximately the same level
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of transcript expression (K4#2, K4#7, K4#10, K4#11), while one has significantly
greater (K4#8) and another significantly less (K4#9) expression of KLK4.
Western blot analysis of KLK4 over-expressing clones, demonstrates that hK4 is
produced and secreted to varying levels by these cells in contrast to the PC-3 native cells
and vector only clones. While not a quantitative assessment, it appears that K4#8
expresses and secretes the greatest amount of protein in accordance with the quantitative
RT-PCR data discussed above. On the other hand, K4#7 also appears to express a
similar quantity of protein as K4#8 as observed on the whole cell lysate blot, but this
does not correlate to the amount of protein secreted or with the quantitative RT-PCR
data. However, studies suggest that in many cell types, the expression of mRNA does
not always parallel protein expression (De Moor and Richter, 1999).
Based on the size of active porcine K4 (doublet at 34 and 37 KDa), the differences in
band sizes between cell lysate and conditioned medium preparations of the hK4 overexpressing cells suggests that pro-hK4 is the protein detected in the intracellular extract
(~38 - 40 KDa), while active hK4 is detected in the conditioned medium (35 KDa). The
hK4 protein may also undergo post-translational glycosylation as the predicted hK4
amino acid sequence contains an N-glycosylation site (Dong et al., 2001). Furthermore,
studies of porcine K4 have demonstrated that the active pig K4 enzyme is glycosylated,
and that deglycosylation was also associated with a loss of proteolytic activity (Ryu et
al., 2002).
Although it was not determined if the hK4 secreted by the cells was enzymatically
active, Western blot analysis of the conditioned medium and subsequent data (see
Chapter 6) indicate that the hK4 over-expressing cells are likely to be secreting active
hK4 protein.
It is possible that PC-3 cells express the putative metalloprotease
suggested to cleave hK4 at the activation site to produce active hK4 (Takayama et al.,
2001). Alternatively, recent studies have shown that hK4 may be auto-activated (Nicole
Willemsen, QUT, personal communication, 2003).
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In addition to the Western blot analysis, immunofluorescence has confirmed that hK4 is
expressed by the transfected clones to varying degrees.
Using both the N and C
terminus hK4 antibodies, the hK4 over-expressing PC-3 clones displayed quite intense
staining in comparison to the native and vector only controls. Negligible staining was
observed in the control cells and in K4#9, which correlates with the quantitative RTPCR data. As the N terminus antibody recognises full length hK4 and variants 3 and 4,
it is likely that the small amount of staining observed in the control cells and K4#9 can
be attributed to endogenous variants 3 and 4.
Similarly, the C terminus antibody
recognises full length hK4 and variant 1, which suggests that the staining observed in the
control cells and K4 #9 when this antibody was used was due to the presence of
endogenous variant 1 protein, since PC-3 cells do not express the full length KLK4
transcript (Chapter 3). With both the N and C terminus hK4 antibody, the greatest
intensity of stain was seen in K4#8 followed by K4#10, K4#11, K4#7, and K4#2.
The predominant cytoplasmic localisation of the protein within the hK4 over-expressing
cells also confirms the presence of exogenous full length hK4. As detailed in Chapter 3,
immunofluorescent staining of LNCaP cells, the only cell line used in this study which
expresses the full length transcript and protein, was primarily cytoplasmic, while those
cells lines which express only the variant transcripts and proteins (RWPE2, DU145 and
PC-3), had a predominant perinuclear localisation within the cell. Accordingly, the
native PC-3 cells and vector only control cells had perinuclear staining indicative of the
exogenously expressed variant proteins.
A significant finding of this study was the stark difference in cell morphology displayed
by the hK4 over-expressing clones when compared with the control cell lines. PC-3
native cells, vector only controls and K4#9 clones were all quite rounded with few
processes and displayed a ‘cobblestone’ appearance when confluent, which is typical of
epithelial cells. In contrast, the remainder of the hK4 over-expressing clones appeared
quite irregular in shape with several processes, suggestive of a more motile phenotype.
Additional phenotypic changes were also apparent. The control cells and K4#9 clones
all adhered well to the culture flask, took considerable time to trypsinise, grew in
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confluent islands and displayed obvious cell-to-cell contacts. The remaining hK4 overexpressing clones did not adhere well to the culture surface and the cells grew
independently of adjacent cells. These findings are intriguing, and may suggest that
increased levels of hK4 influence the morphology and phenotype of the PC-3 cells. PC3 cells have been transfected with various other genes including the androgen receptor
(Dai et al., 1996, Heisler et al., 1997, Snoek et al., 1998, Shen et al., 2000), cell
adhesion molecules (Hsieh et al., 1995) and other serine proteases (Chen et al., 2001) to
observe the effects of these genes on molecular and cellular parameters, yet there is little
mention of altered morphology in these cells or phenotypic changes.
In summary, this chapter reports the successful stable transfection of the PC-3 prostate
cancer cell line of KLK4 into the genome of these cells and the subsequent expression
and secretion of hK4 protein.
The phenotypic changes observed as a result of
KLK4/hK4 over-expression warrant further investigation. Using these cells, the
consequences of hK4 over-expression in a prostate cancer cell line and its role in
prostate cancer cell biology can be examined.
Accordingly, experimentation
characterising the properties of these cells was undertaken to evaluate several biological
functions (such as proliferation, invasion, migration and attachment to extracellular
matrix molecules) important in prostate cancer progression (see Chapter 6).
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CHAPTER SIX
FUNCTIONAL CHARACTERISATION OF
hK4 OVER-EXPRESSING PC-3 CELLS
Chapter 6
6.0 INTRODUCTION
Serine proteases are known to play important roles in proliferation, attachment, matrix
degradation, migration and invasion – key processes associated with cancer metastasis.
Plasmin is one such serine protease which has been implicated in cancer as it is able to
digest ECM molecules and activate latent metalloproteases and growth factors (Frenette
et al., 1997b). Plasmin is formed by the hydrolysis of plasminogen by plasminogen
activators, one of which is urokinase-type plasminogen activator (uPA; Lijnen et al.,
1986). Prostate cancer cells are known to over-express uPA (Achbarou et al., 1994,
Festuccia et al., 1995, Rabbani et al., 1995) which activates plasminogen to generate
plasmin (Lijnen et al., 1986), in turn activating metalloproteases (He et al., 1989,
Stricklin et al., 1977) allowing digestion of ECM proteins, ultimately enabling migratory
cells to escape from the primary tumour site.
Another serine protease implicated in cancer progression, hK2, has been demonstrated to
not only activate uPA, but also to inactivate the endogenous inhibitor to uPA,
plasminogen activator inhibitor-1 (PAI-1) (Frenette et al., 1997a, Takayama et al.,
1997b, Mikolajczyk et al., 1999).
Thus, hK2 may initiate a proteolytic cascade
culminating in plasmin degradation of the ECM and activation of growth factors to
facilitate tumour invasion and proliferation (Frenette et al., 1997b, Takayama et al.,
1997a). hK2 has also been shown to activate the zymogen/pro form of PSA (Kumar et
al., 1997, Lovgren et al., 1997, Takayama et al., 1997a), which in turn has been
implicated in several tumourigenic pathways including increased cellular proliferation
and ECM remodelling and invasion (Liotta and Stetler-Stevenson, 1991, Cohen et al.,
1992, Webber et al., 1995).
Additionally, PSA may be involved in apoptosis,
angiogenesis and bone remodelling (Balbay et al., 1999, Fortier et al., 1999). Clearly,
the processes of invasion and metastasis are complex and involve many different serine
proteases and other factors.
While the importance of both hK2 and PSA in prostate cancer is well established, less is
known about the potential role(s) of KLK4/hK4 in this disease. hK4, like hK2, has been
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implicated in the processes of migration and invasion due to its ability to activate proPSA and single chain urokinase-type plasminogen activator (scuPA, pro-uPA)
(Takayama et al., 2001b). While hK4, PSA and hK2 are only three of the enzymes
associated with prostate cancer, they are potentially capable of degrading the ECM and
activating factors necessary to facilitate tumour migration and invasion. Due to the
similarity of hK4 to porcine enamel matrix serine proteinase 1, which is involved in the
degradation of organic matrix surrounding tooth enamel (Scully et al., 1998; Hu et al.,
2000a; Hu et al., 2000b), it is possible that hK4 also degrades matrix components. It has
been postulated that if the considerable sequence homology between these two enzymes
correlates to function, then the potential exists for hK4 to be involved in prostate cancer
metastasis to bone; the primary site for secondary tumours. Therefore it is important to
determine whether hK4 is capable of interacting with various ECM proteins, in
particular, collagen I, which is the principle protein of bone matrix.
As described above, the putative functional effects of hK4 in prostate physiology and
pathobiology are slowly being revealed; however, the numerous suggested roles of this
enzyme are yet to be experimentally confirmed. The establishment of transfected cell
lines stably over-expressing hK4, as detailed in the previous chapter, will enable specific
roles of hK4 to be identified with respect to various aspects of tumourigenesis and
provides a model to assess the functional roles of hK4 at a cell biology level. To date,
no other study has utilised a prostate cancer model of over-expression to determine hK4
function. Therefore, the aim of this study was to characterise the hK4 over-expressing
cell lines by using a variety of functional assays including proliferation, invasion,
migration and attachment to ECM molecules, to further understand the tumourigenic
properties of this enzyme.
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6.1
6.1.1
MATERIALS AND METHODS
Cell Culture
Stably transfected PC-3 clones were maintained as described in the Materials and
Methods, Section 2.1.1, except that 100µg/ml geneticin (G418) was added to the
medium supplemented with 10% FBS to those clones transfected with pcDNA3.1
expression constructs. As a result, the clones were maintained with constant selection
for cells that have retained the expression vector in their genome. All cells undergoing
functional analysis were passaged approximately 12 h prior to the onset of each assay to
ensure consistency within and between experiments. Specific treatments of the cells for
a range of functional assays are outlined below.
6.1.2
Functional Analysis of hK4 Stably Expressing Clones
As it was not possible to assess the functional characteristics of all clones generated due
to the time consuming nature of the techniques used and expense, the following
experiments were all performed using the same clones for consistency, unless stated
otherwise. Four hK4-expressing clones, K4#8, K4#9, K4#10 and K4#11, were chosen
due to their expression levels of hK4 and changed morphology, as illustrated in the
previous chapter. K4#8 was selected as it had the highest level of hK4 secretion as
determined by western blot analysis (Figure 5.5). K4#10 and K4#11 were chosen as
their cellular morphology and growth in culture were consistent with K4#8. K4#9 was
selected as it clearly expressed KLK4 and hK4, but had a cell morphology dissimilar to
the other hK4 clones (Figure 5.7). Two vector control cell lines (V1 and V3) were
selected for these functional experiments because they clearly did not express high
levels of KLK4/hK4 (see Chapter 5) and they closely resembled the PC-3 parent cell line
with respect to morphology.
6.1.2.1
MTT Tetrazolium Proliferation Assay
The MTT proliferation assay is a widely used assay employed to determine the rate of
proliferation of cultured cells. The principle is based on the reduction of MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to a blue formazan salt by the
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mitochondrial dehydrogenase in viable cells. The method described here is based on the
technique originally developed by Mosmann (1983) with minor modifications.
In
addition to the clones mentioned above, K4#2 and K4#7 clones were also assayed.
Each clone was passaged in T25cm2 flasks at least 12 h prior to the beginning of the
assay to ensure they were in an active proliferating state. Cells that were 80% confluent
or less were subsequently used in the assay. Each clone was washed in PBS, trypsinised
and resuspended in pre-warmed medium containing serum (without geneticin) before
centrifugation at 1000rpm for 5 min. The supernatant was aspirated off the pelleted
cells, which were once more resuspended in medium with serum (1ml). An aliquot of
resuspended cells was removed and diluted in 0.4% trypan blue for cell counting. All
cell counts were performed using a haemocytometer. Five plates of cells were prepared
using 5x103 cells/100µl/well in 24 wells of a 96 well plate and allowed to grow in a
37°C humidified incubator. Each plate was then assayed at various time points (ie 4, 24,
48, 72, 96 h) to determine the rate of proliferation. This involved removing the medium
from each well, and replacing it with 100µl of serum free medium containing 1mg/ml
MTT formazan salt. The MTT solution was left on the cells for 2 h at 37°C, then
removed and replaced with 100µl of DMSO to solubilise the salt. Each day, the medium
on the remaining plates that were not assayed on the day were replaced with fresh serum
free medium. Absorbances were determined spectrophotometrically at 550nm using a
Beckman plate reader.
Results represent an average of three experiments, each
containing 18 replicates of each clone or control.
Statistical analysis on the rate of proliferation (fold increase compared to the “4 h” plate)
of each clone, vector control or parent cell line (calculated with n=3) was performed
using a one-way ANOVA with Tukey’s post-hoc analysis.
6.1.2.2
Preparation of Chemo-Attractants
Chemo-attractants used in chemo-invasion and migration assays include medium
containing 20% FBS and conditioned medium from native PC-3, Saos-2 cells (an
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osteosarcoma cell line with osteoblastic properties) and neonatal foreskin fibroblast
(NFF) cells.
In order to collect conditioned medium from cells, each cell line was grown in the
appropriate medium (PC-3 and NFF: RPMI 1640 medium with 10% FBS; Saos-2:
DMEM with 10% FBS). Once 70-80% confluency was reached, the cells were washed
several times in pre-warmed sterile PBS followed by the addition of 5ml of the
appropriate serum free medium. After 24 h growth, the serum free conditioned medium
was collected, pooled, centrifuged at 1000rpm for 5 min to collect cellular debris, and
the supernatant collected, aliquoted and stored at -20°C until required.
6.1.2.3
Migration (Chemotaxis) Assay
Cell migration was measured by modifying the procedures originally reported by Albini
et al (1987), and the modifications described by Nagakawa et al (1998). Briefly, cells of
interest were added to a migration chamber containing a polycarbonate membrane
through which motile cells can migrate. Figure 6.1 illustrates the basic principles of this
assay. Tissue culture inserts (Falcon), suitable for a 24-well plate format, that were used
in this assay contained polycarbonate membranes with 8µm pores.
Fifty thousand cells (5x104) from each selected clone were harvested and resuspended in
100µl of serum free medium containing 0.1% BSA and added to the upper chamber of
the insert. Five hundred microlitres of chemoattractant (medium containing 20% FBS)
were placed in the chamber of the lower well. Control wells contained 500µl of serum
free medium containing 0.1% BSA. In order to determine the optimal incubation period
for this assay, the preparations were incubated at 37°C for 6, 12, 24 and 48 h in a
humidified atmosphere. Once the correct incubation period was established, a variety of
chemo-attractants was tested, which included conditioned medium from native PC-3
cells, conditioned medium from Saos-2 cells and conditioned medium from NFF cells.
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Proliferating PC-3 cells
Prepare 5x104 cells/100µl/insert
Place chemo-attractant in lower well
50 000 cells
Chemoattractant
Allow migration to proceed for 12 hours
Remove media and non-invaded cells
Fix and stain invaded cells on underside
of membrane
Solubilise stain in acetic acid
Read absorbances at 595nm
Figure 6.1 Migration assay
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In order to quantitate only the cells that had migrated through the membranes, the
medium and non-migrating cells were removed from the upper chamber using a cotton
swab, leaving the migrated cells on the underside of the insert. The migrated cells were
then fixed with 100% methanol for 15 min. Following fixation the cells were stained
with 0.5% crystal violet in 20% methanol for 15 min. To remove excess stain, each
insert was washed under running tap water until the dye ran clear from the insert. Each
insert was allowed to dry for 15 min at room temperature and the stain was solubilised
from the migrated cells in 250µl of 10% acetic acid. Absorbances were read in duplicate
using 2x100µl of the acetic acid solubilised solution at 595nm.
To analyse the results for each clone or control, the absorbance value for each negative
control well for each clone was subtracted from the absorbance obtained from the
respective clone or control’s test chemo-attractant well. This value was divided by the
absorbance obtained for the native PC-3 well and then multiplied by 100%. The result
gave a value which was corrected for background migration and expressed as a final
percentage of the amount of migration in comparison to the native PC-3 cells. Assays
for each clone were performed in duplicate and repeated three times.
As the rate of proliferation of each clone studied was different, as identified by the MTT
proliferation assay, an index for the increase in cell number in response to each chemoattract was calculated. Therefore, the results obtained from the invasion and migration
assays could be corrected for this index to eliminate growth effects. This involved
growing each clone over a 12 h period in each chemo-attractant and determining the rate
of proliferation by MTT formazan salt incorporation, as detailed in Section 6.1.2.1.
Corrected data were analysed by one-way ANOVA and Tukey’s post-hoc analysis.
6.1.2.4
Blocking Assay
In a series of preliminary experiments, two methods of potentially blocking migration
were employed in order to ensure that the migratory effect seen in the clones was a
result of hK4 over-expression.
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6.1.2.4.1
hK4 Antibody Blocking Assay
To block the migratory effect using antibodies, the migration assay (Section 6.2.2.4) was
modified according to the method of Sung and Feldman (2000) by the addition of a
cocktail of kallikrein 4 anti-peptide antibodies. Fifty thousand cells (5x104) were
prepared in media containing 0.1% BSA followed by the addition of 1µl of a 1/50
dilution of hK4 antibody cocktail (equal volumes of N terminal, C terminal and midregion anti-peptide antibodies; see Table 3.4) to the cell suspension.
The cell
suspension/antibody mix was immediately added to a fresh uncoated tissue culture
insert. These inserts were placed into wells containing 500µl of PC-3 cell conditioned
medium chemo-attractant and incubated for 12 h at 37°C. Section 6.1.2.3 details the
remainder of the experiment and method of data analysis.
This experiment was
performed in duplicate. Only the hK4 clones, K4#8 and K4#10, were used in this assay
based on the results of the previous migration assays.
6.1.2.4.2
Aprotinin Blocking Assay
Migration blocking experiments were essentially performed in the same manner as
described in Section 6.1.2.3. However, to block serine protease activity, the serine
protease inhibitor, aprotinin, was added to the cell suspension containing fifty thousand
cells (5x104) in medium with 0.1% BSA. Various concentrations of aprotinin used in
these blocking assays correlated to a range of 0.5 – 5000 kallikrein inactivating units
(KIU). The cell suspension/aprotinin mix was added to a fresh uncoated tissue culture
insert which was then placed in wells containing 500µl of Saos-2 cell conditioned
medium chemo-attractant and incubated for 12 h at 37°C. These experiments were
performed in duplicate and repeated three times. Only the hK4 clones, K4#8 and K4#10,
were used in this assay based on the results of the previous migration assays.
Subsequent steps of this assay and method of analysis are detailed in Section 6.1.2.3.
6.1.2.5
Chemo-Invasion Assay
Chemo-invasion assays are a useful method to determine the invasiveness of many
different cancer cell lines, including the prostate cancer cell line, PC-3 (Hoosein et al.,
134
Chapter 6
1991). Therefore, it was the purpose of this study to determine the invasiveness of the
K4 over-expressing clones and compare their invasive ability to the control cell lines,
PC-3 native and vector-only clones. The clones used in this assay were those stated in
Section 6.1.2.
Invasion experiments were performed in exactly the same manner as the chemomigration assay described in Section 6.1.2.3, except the upper surface of each insert was
coated with growth factor-reduced Matrigel (BD BioSciences) diluted 1:2 in Dulbecco’s
Modified Eagle Medium (DMEM; Life Technologies) in a total volume of 10µl. To
prevent a meniscus of matrigel forming within the insert and to ensure even coverage of
the membrane, 100µl of ice-cold serum free medium containing 0.1% BSA was added
on top of the matrigel layer. The Matrigel-covered inserts were placed at 37°C for 2 h to
allow the gel to set.
Fifty thousand cells (5x104) from each selected clone were harvested and resuspended in
100µl of serum free medium containing 0.1% BSA and added to the upper chamber of
the insert. Five hundred microlitres of chemoattractant (medium containing 20% FBS)
were placed in the chamber of the lower well. Control wells contained 500µl of serum
free medium containing 0.1% BSA. The preparations were then incubated at 37°C for 48
h in a humidified atmosphere. Following incubation, the inserts were fixed, stained and
solubilised as indicated above. Absorbances were determined spectrophotometrically at
595nm. These experiments were performed in duplicate and repeated four times. Data
obtained were corrected against the negative controls and the growth index, expressed in
comparison to native PC-3 cells, and statistical analysis were performed using a one-way
ANOVA and Tukey’s post-hoc analysis.
6.1.2.6 Attachment Assay
Attachment assays were performed to determine whether the over-expression of hK4
might influence the ability of the cells to adhere to various extracellular matrix proteins.
These assays were performed following the procedures reported by Festuccia et al.,
135
Chapter 6
(1999) and Romanov and Goliogorsky (1999). The clones used for this assay were
K4#2, K4#7, K4#8, K4#9, K4#10, K4#11, V1, V3 and PC-3 native cells.
Purified human extracellular matrix molecules, Collagen I, Collagen IV and Fibronectin,
used in these assays were purchased from BD BioSciences. Each matrix molecule was
diluted in PBS to a concentration of 10µg/ml and used to coat 96-well plates to a total
volume of 50µl/ml. After overnight incubation at 4°C, the plates were washed twice in
sterile PBS, followed by 1 h of blocking in 1% BSA in PBS (100 µl/well) at 37°C to
prevent non-specific binding of the cells to any uncoated areas of the wells. In addition,
a separate plate was also prepared with BSA blocking and no coating (“no coat”) of any
matrix molecules. After blocking, the plates were washed three times in sterile PBS.
Preparations of individual clones were resuspended at 2x104 cells/100µl/well followed
by addition to the coated plates and the BSA blocked plate. At this stage, an additional
plate was prepared with cells with no coating and no blocking (“no coat/no block”) to
determine the baseline attachment. The plates were incubated for 1 h at 37°C and then
washed twice to remove any unattached cells, except the “no coat/no block” plate, which
was incubated for 4 h to allow a maximum of cells to attach and was not washed in PBS.
Following the appropriate incubation periods, the cells were fixed in ice-cold 100%
methanol (100µl/well) for 15 min and stained with 0.5% crystal violet in 20% methanol
for 15 min (100µl/well). To remove excess dye, each well was washed several times
then air-dried.
10% acetic acid (100µl/well) was added to solubilise the dye and
absorbances were determined spectrophotometrically at 595nm.
In order to analyse the results for each ECM molecule, the absorbance value for each
clone on the BSA control plate was subtracted from the absorbance obtained from the
coated plate. This value was divided by the absorbance obtained for the “no coat/no
block” plate (ie 100% of cells plated) and then multiplied by 100%. The result gave a
value which was corrected for background attachment to the plate and expressed as a
final percentage of the amount of cells that adhered to plastic (i.e. no coat/no block
136
Chapter 6
plate). Attachment assays were performed in quadruplicate and each experiment was
repeated three times.
137
Chapter 6
6.2
RESULTS
6.2.1 Effect of hK4 Over-Expressing Stably Transfected Cell Lines on the Rate of
Proliferation
All clones generated were assayed for their proliferation rate. These growth rates were
then used to assist in the selection of clones for additional functional analysis. The first
plate (4 h) was assayed on the day of plating to ascertain the original number of cells
plated and attached to each well. Each day thereafter, one plate was assayed every 24 h
and calculated as a fold-increase of the 4 h plate. Figure 6.2A represents the rate of
proliferation for each clone and controls individually. Figure 6.2B represents each clone
type and vector controls, averaged for all replicates.
The degree of proliferation for the combined hK4 clones reveals that there is some
decrease in the growth of the KLK4 transfected cells at 96 h when compared to the
combined vector only controls and PC-3 native cells (Figure 6.2B). Individually, the
results from the proliferation assays show that K4#8 had the slowest doubling time over
the five day period. As determined by the fold change at the 96 h time point, the rate of
proliferation of all the clones from highest to lowest doubling time follows this order:
V3, V1, Native, K4#9, K4#11, K4#10, K4#2, K4#7, K4#8 (Figure 6.2A). Statistical
analysis indicates that all K4 clones grew at a significantly lower rate when compared to
the native PC-3 and vector only controls at 96 h, but there was no statistical difference at
the time points up to and including 72 h.
Having established the proliferation rate for each clone, several clones were chosen for
use in the remaining assays. These clones were selected for several reasons. Each of the
clones were easy to maintain in culture and displayed consistent phenotypes and cell
behaviour.
K4#8 was particularly chosen as its KLK4/hK4 expression at both the
mRNA and protein levels was the highest, and K4#9 was principally selected due to its
phenotype closely resembling the native and vector only control cells, along with having
a similar expression level of KLK4 to the control cells at the mRNA level.
138
Chapter 6
A.
8
Native
7
V1
Fold Increase
6
V3
5
K4 #2
4
K4 #7
3
K4 #8
K4 #9
2
K4 #10
1
K4 #11
0
4 hr
B.
24 hr
48 hr
72 hr
96 hr
8
7
Fold Increase
6
5
*
4
Native
Vectors
K4 clones
3
2
1
0
4 hr
24 hr
48 hr
72 hr
96 hr
Figure 6.2. Rate of proliferation assessed using the MTT tetrazolium proliferation
assay.
Both panels represent mean data generated from three separate experiments, each
containing 18 replicates for each cell/clone type. Panel A represents the fold increase
over the 4 h control (assigned a value of 1) up to 96 h of growth for the PC-3 native,
individual vector only clones (V1, V3) and individual K4 over-expressing clones (K4#2,
K4#7 - #11). Panel B is a summary graph of the data in Panel A averaged for each clone
type. Standard errors of the mean are indicated with bars.
Statistical significance
(calculated using n=3) (denoted by an asterix) was obtained using a one-way ANOVA
using Tukey’s Post-hoc analysis (p < 0.01).
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Chapter 6
6.2.2 Effect of hK4 Over-Expressing Stably Transfected Cell Lines on Cell Motility
Figure 6.3 presents the results obtained from the chemotaxis/migration experiments
carried out to determine the optimal incubation period for the assay. The result from the
PC-3 native cells was set at 1, each clone was corrected against its respective negative
control well and all results were normalised to take proliferation into account over each
time period in 20% FBS. Each experiment was performed in duplicate three times with
the exception of the 6 h assay which was performed only once.
While statistical significance was not achieved for the 12 or 48 h assays, the 24 h
experiment resulted in K4#8 and K4#10 exhibiting a significant increase in migratory
ability towards the chemo-attractant over and above the native PC-3 cells and vector
only controls. K4#9 and K4#11 displayed a similar capacity to migrate through the
membrane as did the native and vector only cells. Interestingly, the results of the 12 h
experiment closely resemble those of the 24 h assay for each clone; however the result
was not significant due to the fold change being much lower.
The 12 h time point was chosen for all subsequent migration assays, as it was obvious
upon examination of the lower chamber of the 24 and 48 h experiments that these assays
had gone to completion since many migrating cells had plated onto the surface of the
lower chamber. Once cells release from the underside of the membrane and seed into
the lower chamber, the assay no longer accurately reflects the number of cells migrating
through the pores of the membrane. Migration assays are typically performed between 5
and 12 h depending on the size of the migrating cell and the size of the membrane’s
pores (Djakiew et al., 1993).
Once the 12 h time point was established as optimal, chemotaxis/migration assays were
performed utilising various chemo-attractants to determine whether the hK4 clones
preferentially migrate towards soluble factors secreted from particular cell types. Figure
6.4 presents the cell motility of the hK4 clones towards four different chemo-attractants
over 12 h (n=3, performed in duplicate). The results from the PC-3 native cells were
again set at 1 for ease of comparison, each clone was corrected against its respective
140
Chapter 6
A.
6 hr
Fold Change
1.5
1
0.5
0
Native
V1
B.
V3
K4 #8
K4 #9
K4 #10
K4 #11
K4 #9
K4 #10
K4 #11
12 hr
Fold Change
2.5
2
1.5
1
0.5
0
Native
V1
V3
K4 #8
24 hr
Fold Change
C.
6
5
4
3
2
1
0
*
*
Native
V1
D.
V3
K4 #8
K4 #9
K4 #10
K4 #11
K4 #9
K4 #10
K4 #11
Fold Change
48 hr
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Native
V1
V3
K4 #8
Figure 6.3 Migratory potential for hK4 clones at various time points.
Histograms of the fold change for migration experiments through an 8 µm pore size
membrane barrier over 6, 12, 24 and 48 h using 20% FBS as a chemo-attractant, for hK4
over-expressing cells (K4 #8-11) when compared to PC-3 native and vector only
controls (V1, V3) (n=3 experiments, performed in duplicate). Standard errors of the
mean are indicated with bars. Statistical significance is indicated by an asterix (p<0.05).
141
Chapter 6
A.
20% FBS
Fold Change
4
3
2
1
0
Native
V1
V3
K4 # 8 K4 # 9 K4 # 10 K4 # 11
PC-3 Conditioned Media
B.
Fold Change
10
*
8
*
6
*
4
2
0
Native
C.
V1
V3
K4 #8
K4 #9 K4 #10 K4 #11
Saos2 Conditioned Media
Fold Change
4
*
*
K4 #8
K4 #9 K4 #10 K4 #11
3
2
1
0
Native
V1
V3
NFF Conditioned Media
D.
Fold Change
4
3
2
1
0
Native
V1
V3
K4 #8
K4 #9
K4 #10
K4 #11
-1
Figure 6.4 Migratory potential of hK4 clones towards various chemo-attractants.
Graphs of the fold change for migration experiments through a membrane barrier over
12 h towards 20% FBS and conditioned medium from PC-3, Saos-2 and NFF cells, for
hK4 over-expressing cells (K4 #8 - 11) when compared to PC-3 native and vector only
controls (V1, V3) (n=3 experiments, performed in duplicate). Standard errors of the
mean are indicated with bars. Statistical significance is indicated by an asterisk (p<0.05).
142
Chapter 6
negative control well in each chemo-attractant and all results were normalised to take the
rate of proliferation over a 12 h period into account.
As noted above, the ability of the hK4 clones to migrate towards 20% FBS was not
significant (Panel A), although a trend was observed with K4#8 and K4#10 displaying
increased migration and K4#9 and K4#11 exhibiting decreased migration.
However,
when conditioned medium from native PC-3 cells was used as a chemo-attractant, three
of the four hK4 clones examined demonstrated a significant increase in motility when
compared to the native and vector only cells (Panel B). Most strikingly, K4#8 had a 7
fold greater ability to migrate than the native cells, followed by K4#10 with a 6.4 fold
increase and K4#11 with a 4.5 fold increase in migratory potential. Interestingly, K4#9,
which has a similar morphology to the native cells (Figure 5.8) and much lower levels of
KLK4 (Figure 5.4) compared with the other KLK4 over-expressing cells, did not exhibit
significantly different migratory ability to the native or vector only cells.
A similar pattern of migratory potential of the hK4 clones was seen when their motility
towards conditioned medium from the osteoblast-like cell line, Saos-2, was assessed
(Panel C). In this experiment, K4#10 had the greatest motility with a 2.9 fold increase
over the native cells, followed by K4#8 with a 2.7 fold increase and K4#11 with a 2.4
fold increase.
Again, K4#9 proved to be less motile than the native PC-3 cells.
Statistical significance was achieved only for the K4#8 and K4#10 clones.
Finally, conditioned medium from NFF cells was used as a chemo-attractant in order to
determine whether the hK4 clones’ migratory potential was increased by the soluble
factors of the two particular cell lines used (prostate cancer and bone related) or as a
function of soluble factors found in conditioned media from a non-epithelial cell line i.e.
fibroblast and non prostate cancer related line. The results (Panel D) show that the hK4
clones do not exhibit significantly increased migration towards this chemo-attractant,
with all hK4 clones displaying less than a 1.6 fold change over the native PC-3 cells.
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Chapter 6
6.2.3 Effect of hK4 antibodies on hK4 Mediated Cell Motility
In order to test the specificity of the migratory effects of K4#8 and K4#10 towards PC-3
conditioned medium, in a preliminary experiment, a mixture of three hK4 anti-peptide
antibodies was added to the cell suspension prior to the onset of the migration assay. At
a dilution of 1:50, the hK4 antibodies appeared to decrease the K4#8 cells effect on cell
motility but the antibody blocking effects appeared marginal at best (Figure 6.5).
Further antibody blocking experiments were not pursued due to the lack of readily
available supplies of the hK4 antibodies and lack of evidence that these antibodies were
in fact hK4-function blocking antibodies.
6.2.4 Effect of Aprotinin on hK4 Mediated Cell Motility
In order to test whether the serine protease activity of hK4 was responsible for the
increase in migratory potential of the hK4 over-expressing cells, aprotinin, a serine
protease inhibitor, was added to the cell suspension prior to the onset of the migration
assay. This experiment was performed in an attempt to block the migratory effects of
K4#8 and K4#10 towards Saos-2 conditioned medium. Figure 6.6 presents the cell
motility of the hK4 clones in the absence and presence of an increasing concentration of
aprotinin (100-1000 Kallikrein Inactivating Units (KIU)) over 12 h (n=3, performed in
duplicate). The results from the PC-3 native cells were again set at 1 for ease of
comparison, each clone was corrected against its respective untreated control well and
all results were normalised to take into account the rate of proliferation over a 12 h
period in Saos-2 conditioned medium.
Firstly, as expected, both K4#8 and K4#10 exhibited significantly greater motility when
compared with the native and vector only cells. However, in this group of experiments,
K4#8 had a 4.4 fold increase over the native cells, which is greater than the fold change
observed previously (Figure 6.4, Panel C). The fold change of K4#10 in this assay was
comparable to that seen previously (Figure 6.4, Panel C) with a 2.9 fold increase in
migration over the native cells.
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Chapter 6
hK4 Antibody (1:50)
Fold Change
12
10
8
6
4
2
0
Native Native
+Ab
V1
V1 +Ab K4 #8
K4 #8 K4 #10 K4 #10
+Ab
+Ab
Figure 6.5 Effect of hK4 antibodies on hK4 mediated cell motility.
Graphical representation of the fold change for migration experiments in the presence of
hK4 anti-peptide antibodies at 1:50 dilution (n = 1 experiment, performed in duplicate).
The chemoattractant used in this assay was PC-3 conditioned medium. The results for
the PC-3 native cells were set at a value of 1 for ease of comparison and each clone is
indicated as a fold-increase of this value.
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Chapter 6
6
5
3
1
K4 #10 + 250 KIU
K4 #10 + 100 KIU
K4 #10
K4 #8 + 1000 KIU
K4 #8 + 500 KIU
K4 #8 + 250 KIU
K4 #8 + 100 KIU
K4 #8
V1 + 1000 KIU
V1 + 500 KIU
V1 + 250 KIU
V1 + 100 KIU
V1
N + 1000 KIU
N + 500 KIU
N + 100 KIU
Native
-1
N + 250 KIU
*
0
*
*
*
*
K4 #10 + 1000 KIU
2
K4 #10 + 500 KIU
Fold Change
4
Figure 6.6 Effect of aprotinin on hK4 mediated cell motility.
Graphical representation of the fold change for migration towards Saos-2 conditioned
medium in the presence of increasing concentrations of aprotinin (with corresponding
kallikrein inhibitor units indicated (100 - 1000)) (n = 3 experiments, performed in
duplicate). The results for the PC-3 native cells were set at a value of 1 for ease of
comparison and each clone is indicated as a fold-increase of this value. Standard errors
of the mean are indicated with bars. Statistical significance is indicated by an asterisk
(p<0.05).
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Chapter 6
In the presence of aprotinin, the migratory potential of the hK4 clones was markedly
decreased at all concentrations of kallikrein inhibitor units (KIU) examined. Although
not statistically significant, the migration of K4#8 in the presence of 100 KIU was half
that of K4#8 without aprotinin, and in the presence of 250, 500 and 1000 KIU, the
migration of this clone was less than one quarter. A similar pattern was observed with
K4#10. In the presence of 100 KIU, the number of K4#10 cells that migrated through
the membrane was half that of its corresponding control. Again, in the presence of 250,
500 and 1000 KIU, the migration of this clone was less than one quarter of K4#10 in the
absence of aprotinin. The values for 500 and 1000 KIU were statistically significant for
K4#10.
Although the initial migration of the control cells was not as marked as with the hK4
clones, the migration of the native PC-3 and vector only control cells were also inhibited
in the presence of aprotinin. This was not surprising due to the presence of other
proteases endogenously expressed in the PC-3 cells which would have been inhibited by
aprotinin. Statistical significance was reached at 250 and 500 KIU for the native cells
and at 500 KIU for the vector only control cells.
6.2.5
Effect of hK4 Over-Expressing Stably Transfected Cell Lines on Cell
Invasion
The invasive ability of the hK4 over-expressing cell lines towards 20% FBS was
examined using Matrigel-coated membranes over 48 h and compared to that of the PC-3
native cell line and vector only controls (Figure 6.7). The experiments were performed
in duplicate on three separate occasions. Each clone was corrected against its respective
negative control well and assayed for the proliferation rate over this 48 h period in 20%
FBS in order to correct for changes in the growth index. The PC-3 native cell line was
set at 1 for ease of comparison.
Three of the four hK4 over-expressing clones (K4#8, K4#10 and K4#11) showed a
slight increase of 20% in invasive potential over that of the parent cell line and an
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Chapter 6
1.8
1.6
Fold Increase
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Native
V1
V3
K4 #8
K4 #9
K4 #10
K4 #11
Figure 6.7 Invasive potential for hK4 over-expressing clones compared to controls.
Graphical representation of the fold change for invasion assays through a Matrigelcoated membrane barrier for hK4 over-expressing cells (K4 #8 - 11) when compared to
PC-3 native and vector only controls (V1, V3) over 48 h (n = 3 experiments performed
in duplicate). The results for the PC-3 native cells were set at a value of 1 for ease of
comparison and each clone is indicated as a fold-increase of this value. Standard errors
of the mean are indicated with bars. Statistical analysis using a one-way ANOVA with
Tukey’s Post-hoc analysis was performed, however all cells exhibited non-significant
changes in invasive potential.
148
Chapter 6
increase in 10 - 20% when compared to the vector only controls, although this was not
statistically significant. K4#9 exhibited a 5% decrease in invasive ability compared to
PC-3 native and V3 and a 15% decrease when compared to V1. Again, this result was
not statistically significant.
An invasion assay was also performed (n=1, performed in duplicate) using Saos-2
conditioned medium as the chemo-attractant at both 24 and 48 h. No invasion of any of
the cells examined was observed (data not shown), and therefore invasion assays were
not pursued further.
6.2.6 Effect of hK4 Over-Expressing Stably Transfected Cell Lines on Attachment
to Extracellular Matrix Molecules
Attachment assays using matrix coated plates give an indication of a cell’s ability to
adhere to different substrates over a 1 h period at 37°C. Each clone was assayed in
quadruplicate and each experiment was repeated 3 times. Fibronectin, Collagen I and
Collagen IV were chosen as these three matrices are found in different areas of the ECM
which migrating cells would traverse during the metastatic process. Collagen IV is found
in the basement membrane, fibronectin in the stroma, and collagen I in the bone
interstitial matrix.
When the attachment properties of the cells to fibronectin, an ECM molecule
predominantly found in stromal tissue, were examined, no significant differences were
seen for any of the hK4 over-expressing clones compared to the PC-3 native and vector
only control cell lines (Figure 6.8).
Analysis of the cell’s ability to attach to collagen IV, an ECM molecule found in the
basement membrane, found that two of the hK4 clones (K4#10 and K4#11) exhibited
significantly greater attachment than the native and vector only cells, while two other
hK4 clones (K4#7 and K4#9) exhibited significantly less attachment. The hK4 clones,
K4#2 and K4#8, demonstrated no significant differences in their ability to attach to
collagen IV when compared to the native PC-3 cells and the vector only controls.
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Chapter 6
Fibronectin
Percent of Control
60
50
40
30
20
10
0
Native
V1
V3
K4 #2 K4 #7 K4 #8 K4 #9 K4 #10 K4 #11
Collagen IV
Percent of Control
60
*
50
40
*
30
*
20
*
10
0
Native
V1
V3
K4 #2 K4 #7 K4 #8 K4 #9 K4 #10 K4 #11
Collagen I
Percent of Control
60
*
50
*
40
*
30
*
*
20
10
0
Native
V1
V3
K4 #2 K4 #7 K4 #8 K4 #9 K4 #10 K4 #11
Figure 6.8 Percent attachment of hK4 over-expressing clones and control cell lines
to the extracellular matrix molecules Fibronectin, Collagen IV and Collagen I.
Graphical representation of the cellular attachment of hK4 over-expressing cells (K4 #8
- 11) to Fibronectin (Panel A), Collagen IV (Panel B) and Collagen I (Panel C) when
compared to PC-3 native and vector only controls (V1, V3) as a percent of total cell
number plated over 1 h at 37°C (n=4 replicates, 3 experiments). Standard errors of the
mean are indicated with bars. Statistical significance is denoted by an asterisk (p<0.01).
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Chapter 6
Interestingly, hK4 over-expressing clones, K4#2, K4#8, K4#10 and K4#11, were able to
adhere more readily to collagen I, the predominant interstitial matrix molecule in bone,
over and above the native PC-3 cells and vector only controls. Again, K4#9 exhibited a
significant decrease in adhesive ability.
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Chapter 6
6.3 DISCUSSION
In order to metastasise, cells of the primary prostatic tumour must possess mechanisms
rendering the cells capable of degradation of components of the extracellular matrix,
adhesion to and degradation of the underlying basement membrane, intravasation into
blood or lymph vessels, and subsequent extravasation and proliferation in a specific,
secondary organ.
These stages are governed by characteristics of the cellular
environment and those inherent to the tumour cell. Of those factors intrinsic to the
tumour cell, these include, but are not limited to, hormone receptor status, expression of
cell adhesion molecules, increase in motility and the production of proteolytic enzymes
(Liotta, 1986; Kohn and Liotta, 1995). Prostate cancer cells have been found to express
the proteolytic enzyme, hK4, at significantly greater levels than cells from a benign
prostate (see Chapter 3). This study has shown that cells which over-express hK4
participate in various pathways associated with invasion and metastasis. Specifically,
hK4 over-expression has influenced cell motility and the adhesive properties of PC-3
prostate cancer cells.
No previous studies have examined the function of hK4 in an in vitro over-expression
system. This study has found that when hK4 is introduced into PC-3 cells, its expression
decreased the proliferation (2 fold) when compared to the PC-3 native cells at 96 h. If
hK4 were an activator of growth factors or growth factor availability, like other
kallikreins, such as PSA, which cleaves the IGFBP-3/IGF complex releasing bioactive
IGF (Cohen et al., 1992), then it would be expected to increase cellular proliferation.
However, the results of this study demonstrated an overall decrease in proliferation
suggesting that hK4 does not have a role in increasing stimulatory growth factors.
Alternatively, hK4 could potentially cause the activation of factors that inhibit
proliferation, such as TGFβ, or it could activate inhibitors of mitogenic factors which
would in turn result in a decrease in cellular proliferation.
It is also possible that the
reasons behind this slower growth rate may lie with the amount of hK4 produced by the
transfected cells. This has been demonstrated in the previous chapter where it was
shown that K4#8 secretes the greatest quantity of hK4, and, of all the hK4 clones tested,
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Chapter 6
had the slowest growth rate.
This implies that hK4 may elicit a dose responsive
decrease in cellular proliferation.
This action may be via the direct induction of
apoptosis, or in an in vivo setting, it may act indirectly, through its activation of PSA
which is suggested to be involved in inducing programmed cell death (Balbay et al.,
1999). Alternatively, as the activity of hK4 in the cells is largely unknown since an
enzyme assay for hK4 activity is not available, these results may also be indicative of a
lack of enzymatically active hK4.
If the cells do not express the putative
metalloprotease suggested to cleave hK4 at the activation site to produce active hK4
(Takayama et al., 2001), then any ability hK4 may have to activate mitogenic growth
factors would be lost. However, other enzymes may be present in PC-3 cells that
activate pro-hK4 or it may be auto-activated (Nicole Willemsen, personal
communication, 2002).
While malignant cells proliferate in the primary tumour, they gain the necessary
parameters required for invasive potential. These include, recognition and adherence to
ECM components, initiation of the necessary proteases required to degrade the ECM and
subsequent migration through the disrupted matrix (Wells, 2000; Kassis et al., 2001).
Cell migration has been suggested to be the rate limiting step in cancer cell invasion and
metastasis (Kasssis et al., 2001) and was therefore an important parameter assessed in
this study.
As shown in this chapter, hK4 over-expressing clones have increased motility/migration
across a synthetic membrane barrier when compared with the control clones over
various time points and in response to a range of different chemo-attractants. Over a
twelve h period, the hK4 clones displayed an increase in migration towards the soluble
factors produced by prostate (PC-3) and bone (Saos2) cells, but not towards soluble
factors from normal fibroblasts (NFF). This indicates that the increase in migration
observed towards the prostate and bone conditioned media was specific to those
particular cell types and not due to factors characteristic of conditioned medium from
any cell type.
There was a greater increase in migration observed when PC-3
conditioned medium was used as a chemo-attractant than that from the osteoblast-like
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cell line. This perhaps reflects a system whereby prostatic soluble factors have a greater
capacity to influence K4 induced migration. Interestingly, the hK4 clones were also able
to migrate towards soluble factors from Saos2 cells supporting other studies which
suggest that prostate cancer cells preferentially disseminate to bone
(Hujanen and
Terranova, 1985, Jacob et al., 1999, Bubendorf et al., 2000, Rubin et al., 2000).
Furthermore, the PC-3 cell line was initially cultured from a lumbar vertebrae bone
metastasis of a poorly differentiated prostatic adenocarcinoma, (Kaighn et al., 1979). It
has been demonstrated that bone extracts are potent chemoattractants over extracts from
other tissues and that they increase migration and invasion by prostate cancer cell lines
(Jacob et al., 1999). Many of these soluble factors, which include TGFβ, bFGF, KGF,
IGFs and bone morphogenic proteins, also stimulate the growth of metastatic cancer
cells in the marrow (Hauschka et al., 1986).
Migration assays performed in the presence of a cocktail of hK4 anti-peptide antibodies
did not result in a decrease in migratory potential. One likely reason for this is because
the antibodies are not “blocking antibodies”, that is, the antibody epitopes are not at or
near the active site of the enzyme. Alternatively, it may be that insufficient antibody
was used to result in a marked decrease. However, migration assays performed in the
presence of the serine protease inhibitor, aprotinin, did reveal a decrease in the migratory
potential of the hK4 clones at all inhibitor concentrations examined. Studies utilising
recombinant hK4 (rhK4) have shown that rhK4 is inhibited by aprotinin by forming a
27kDa complex with it (Takayama et al., 2001). Considering the findings of Takayama
and colleagues, it suggests that the increased migration exhibited by the K4 clones is in
fact due to the over-expression of hK4.
In order to become invasive and metastasise, malignant cells must detach from the
primary tumour and interact with the surrounding molecules of the basement membrane,
stroma and structures of the endothelium. Cell migration and invasion are regulated by
the adhesive interactions between cell surface molecules and ECM proteins. These
interactions must be sufficient enough to generate the needed traction along the
substratum, but weak enough to prevent permanent adhesion (Huttenlocher et al., 1995)
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as excessive adhesion may result in disabled detachment and movement. In order to
study the adhesive properties of the hK4 over-expressing cells, attachment assays,
involving various ECM and BM components, were performed.
This study demonstrated that hK4 over-expressing cells displayed no increase over
control cells in their attachment to fibronectin; however, some hK4 clones showed
increased adhesion to Collagen type IV, while five hK4 clones exhibited increased
attachment to Collagen I. It is possible that K4, being a trypsin-like enzyme, is involved
in the process of cellular attachment via activation of protease-activated receptors
(PARs), a subfamily of G-protein-coupled receptors. Interestingly, trypsin is known to
activate PAR2 and PAR4 resulting in the activation of members of the Rho family
which are involved in cytoskeletal reorganisation. In particular, PAR2 has been shown
to be the primary mediator of RhoA activation which also causes stress fibres and focal
adhesions of prostate cancer cells (Greenberg et al., 2003). PAR activation is also
associated with increased cell adhesion to matrix proteins, secretion of MMPs and
increased cell motility. Considering the ability of the hK4 clones to adhere to the matrix
proteins, Collagen type I and IV, and their increased motility over the control cells, it is
possible that the mechanism allowing these actions is via hK4-mediated activation of
one or more of the PARs. Furthermore, PAR1 has been shown to have increased
expression in prostate cells derived from bony metastases (PC-3 and VCaP) and has
been implicated in the early stages of prostate cancer metastasis (Cooper et al., 2003).
As Collagen I is the major constituent of the bone interstitial matrix and all but two of
the hK4 clones exhibited an increased attachment to this protein, then it is possible that
hK4 plays an important role in prostate cancer metastasis to the skeleton.
Considering the hK4 clones displayed increased motility towards soluble factors from
bone cells and an increased attachment to the bone matrix protein collagen I, it is
possible that hK4 may play a key role in the tendency for prostate cancer cells to
preferentially disseminate to bone. This is particularly important as up to 90% of
patients with advanced prostatic cancer have bone metastases (Bubendorf et al., 2000,
Rubin et al., 2000). Bone metastasis is generally associated with a poor prognosis as the
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growth rate of the secondary tumour in bone marrow is considerably greater than that of
the slowly growing primary prostatic tumour (Berrettoni et al.,1986). Not only does it
cause considerable pain and suffering to the patient, it also indicates that the malignant
process is incurable (Scher & Chung, 1994).
The development of new and effective
therapeutic treatments for the management of late stage prostate carcinoma, of which
hK4 may be a candidate target, depends therefore on a better understanding of the
mechanisms that underlie the predilection of this malignancy to bone.
Many of the hK4 clones also displayed altered morphology and phenotype in
comparison with the native and vector only cells, with an increased number of processes
and an irregular appearance.
Furthermore, the hK4 clones also demonstrated
compromised attachment to the growth surface, whereas the native and vector only cells
did not exhibit these characteristics (see Chapter 5). It is commonly accepted that
epithelial cells that appear spindle-shaped are more likely to display an invasive/motile
phenotype, as often reported in other epithelial cancers, and this trend is confirmed here
(Sommers et al., 1992; Boyer et al., 1996). The phenotypic alterations observed in the
hK4 over-expressing cells may be a result of epithelial-mesenchymal transition (EMT).
Essentially, this transition is associated with various characteristics displayed by the hK4
cells; including cell dispersion/scattering, disruption of intercellular junctions, and the
acquisition of increased cell motility (Boyer et al., 1996). The transition from epithelial
to mesenchymal phenotypes is thought to be linked to the early steps of invasion and
metastasis (Hay, 1995).
There is much evidence demonstrating that PC-3 cells are among the most invasive of
the prostate cancer cell lines (Hoosein et al., 1991), and experimentation in vivo has
demonstrated that it is the most metastatic prostate cancer cell line (Kaighn et al., 1979;
Shevrin et al., 1989; Rembrink et al., 1997). Therefore, it was the aim of this study to
determine whether hK4 had an influence over the invasive capabilities of the PC-3 cell
line, in order to determine a possible role for hK4 in tumourigenesis.
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Analysis of the in vitro invasive potential for the hK4 clones revealed no statistically
significant increase in invasion over the control clones.
This was a somewhat
unexpected result considering their spindle shaped morphology, increased motility and
the fact that active hK4 is known to activate pro-uPA; all processes which may enhance
invasion (Takayama et al., 2001). However, as invasion involves destruction of the
basement membrane (BM) and migration requires only destruction of the ECM around
the migrating cell, it is possible that although the hK4 clones possess the ability to
migrate and therefore potentially destroy ECM components, they may not possess the
mechanisms capable of degrading the BM. The BM consists mainly of collagen type
IV, heparan sulfate proteoglycan, laminin, and sometimes fibronectin. Interestingly, the
degradation of type IV collagen in the BM is believed to be the crucial step when a cell
invades the BM (Albini, 1998). Although the results indicate that hK4 may not be
driving the cell towards an invasive phenotype, the Matrigel model that is often used
does not take into account the many complicated interactions present in a whole
organism (Wells, 2000). Furthermore, the major components of Matrigel are laminin,
collagen IV, entactin and heparan sulfate proteoglycan (Kleinman et al., 1982).
Notably, collagen type I is absent from the matrigel components, and given that the K4
cells attach preferentially to this ECM protein, the lack of it may account for the
inability of the cells to invade. Additionally, several studies have shown that Matrigel
does not provide a universal model to correlate the invasiveness of cells in vivo and in
vitro (Noel et al., 1991, Simon et al., 1992, Manske and Bade, 1994) and inconsistencies
across batches of Matrigel have been reported to skew results (Janiak et al., 1994).
Therefore, it is important that animal studies be conducted with these clones to
definitively determine their effect on cellular invasion as the “gold standard” for
invasion studies relies on histopathologically identified invasion of tumours in a living
host (Wells, 2000).
In summary, it appears that hK4 may be important in various pathways leading to the
progression of prostatic carcinoma, although the precise mechanism(s) by which it is
involved is yet to be determined (Figure 6.9).
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hK4
uPA
Plasmin
PARs
MMPs
Degradation of
ECM
Increased cell
motility
Rho Family
Increased
adhesion to
ECM
Morphological
changes
Prostate cancer
progression
Figure 6.9 Potential Mechanisms of hK4 Action
hK4, a trypsin-like enzyme, may contribute to prostate cancer progression by its
potential involvement in the processes of motility, cellular attachment and changed
morphology via the activation of PARs, a subfamily of G-protein-coupled receptors. It
may cause increased cellular motility indirectly via the uPA pathway and may act
directly in the process of ECM adhesion to facilitate progression of the tumour.
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The evidence presented here suggests that hK4 may facilitate tumour cell migration, via
modification of cellular morphology and attachment to Collagen type I and IV, key
components of the extracellular matrix. Although it was not determined if the hK4
secreted by the cells was enzymatically active, the partial blocking of migration via the
serine protease aprotinin, is compatible with active hK4 as a causative agent of
migration of these cells.
Therefore, the data presented suggests that the altered
phenotype, the increased migratory potential of the cells and the enhanced attachment to
certain ECM molecules supports the theory that hK4 is involved in prostate tumour
progression.
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CHAPTER SEVEN
GENERAL DISCUSSION
Chapter 7
7.0 INTRODUCTION
This thesis has aimed to (1) characterise the expression patterns of KLK4 transcripts in
prostate cancer and benign prostatic hyperplasia (BPH) to determine whether they may
play an important role as new biomarkers for this disease; (2) to extend the expression
profile of KLK4 and variant transcripts in a range of prostate cancer cell lines
representing a disease spectrum from androgen dependent to metastatic androgen
independent phenotypes; (3) to broaden the known profile of hormones and growth
factors which regulate KLK4 expression in an androgen dependent prostate cancer cell
line; and (4) to advance the current understanding of the functional role of hK4 in the
progression of prostate cancer.
7.1 KLK4/hK4 Transcripts in Prostate Tissue Samples and Prostate Cancer Cell
Lines
The underlying principle of the first part of the project was based on the current clinical
use of other members of the kallikrein family in the diagnosis and monitoring of prostate
cancer. PSA and, more recently, KLK2, have proven extremely useful in detecting and
monitoring prostate disease. However, neither one can discriminate between prostate
cancer and BPH with high specificity. Hence it was thought that KLK4, which is known
to be highly expressed in the prostate, may be another useful biomarker of prostate
disease. This study aimed to determine whether KLK4 may act as a more specific
marker in detecting prostate cancer as opposed to BPH.
While the use of the PSA screening test has dramatically changed the way physicians
detect and subsequently treat men with prostate cancer, there are known difficulties
associated with relying on elevated PSA levels as a definitive marker of cancer. A key
issue in cancer diagnosis is that men suffering from benign forms of disease such as
BPH can present with clinically elevated concentrations of PSA, with virtually all men
over the age of 50 having histological evidence of BPH. Furthermore, while PSA has
relatively high sensitivity, detecting at least 75% of men with cancer, specificity is
problematic with up to 30% of men with elevated PSA having no evidence of cancer
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(Rittenhouse et al., 1998, Brawer, 2000, Thompson et al., 2003).
Therefore the
usefulness of PSA as a marker for prostate cancer has been challenged and has led to the
development of new applications based on the original PSA test (Stephan et al., 2002,
Haese et al., 2003). These applications include the use of different forms of PSA (‘free’
PSA and PSA bound to inhibitors) which has improved the value of PSA testing in men
suspected of having prostate cancer (Brawer, 2000). Nevertheless, these applications
have only been partially successful in the discrimination between benign and malignant
disease, and research concentrating on finding PSA and hK2 variants which may be
more specific and sensitive markers for malignancy has increasingly been reported,
(Mikolajczyk et al., 1997, Herrala et al., 1998, Mikolajczyk et al., 2000a, Mikolajczyk
et al., 2000b, Mikolajczyk et al., 2000c, Mikolajczyk et al., 2001, Peter et al., 2001,
Mikolajczyk et al., 2002).
The results of this study using real-time RT-PCR for “total” KLK4 (which includes the
full length transcript and four variant transcripts) using samples from both prostate
cancer and BPH patients revealed differential expression for “total” KLK4. Levels of
KLK4 transcripts were elevated in prostate cancer specimens, compared with patients
afflicted with benign disease. Although the samples were obtained from heterogeneous
tissue populations, the findings gained from this study still show a clear increase in
KLK4 expression in cancer tissues. Real-time RT-PCR for PSA using the same tissue
samples also revealed an increase in the cancer over the benign samples, although the
increase observed in KLK4 expression was 2.5 times greater than that observed for PSA.
While this initial finding appears very promising, further investigation to confirm this
result using a larger patient cohort is essential. Additionally, within that cohort, greater
representation of each grade and stage of prostate cancer is necessary in order to
determine if a relationship exists between specific histopathological stages of prostate
cancer and KLK4 transcript copy numbers. Clinical follow-up data is also necessary to
gain information as to whether KLK4 transcript copy numbers may be useful as a
prognostic indicator.
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Not only is an increased number of tissue samples necessary, but laser capture
techniques producing a homogeneous population of cells (Rubin, 2001) would be
valuable in confirming these findings. Laser capture microdissection (LCM) is a
relatively new technique which allows examination of a selected and pure population of
cells which is significantly more informative than molecular analysis performed on bulk
tissue samples. LCM has now been used successfully for numerous studies in the field
of prostate cancer research (Ornstein et al., 2000a, Ornstein et al., 2000b, Simone et al.,
2000, Rubin et al., 2000a).
A number of kallikreins have variant mRNA transcripts which appear to be expressed
differentially or preferentially in malignant tissues (Henttu et al., 1990, Baffa et al.,
1996, Hilz et al., 1999, Mikolajczyk et al., 2000b, Tanaka et al., 2000, Yousef et al.,
2000, Chang et al., 2001, Dong et al., 2001, Korkmaz et al., 2001, Magklara et al., 2001,
Mikolajczyk et al., 2001, Dong et al., 2003).
This study has contributed to the
knowledge of the expression of “total” KLK4 mRNA transcripts in prostate disease, but
it is now important to determine which of these transcripts may be of greatest
importance. However, given it is not possible to design primers to detect each variant in
isolation, it may only be possible to further characterise the full length transcript as
distinct from the combined variants when employing an RT-PCR based approach.
A potential clinical use for the KLK4 transcripts may be to use an RT-PCR based
strategy for analysis of biopsy tissues, circulating cancer cells or shed cancer cells in
urine and/or ejaculate. Once a transcript concentration range has been established for a
given gene transcript, quantitative RT-PCR for KLK4 may provide a way of determining
whether a patient has prostate cancer in addition to histopathological analysis of the
biopsy sample. As RT-PCR is a powerful and highly sensitive tool that has the ability to
detect small numbers of prostatic cells disseminated within the peripheral blood or
within other body fluids or tissues, an RT-PCR based strategy may be particularly useful
to detect early relapse. One previous study has correlated KLK2 mRNA levels in
circulating cancer cells with aggressive phenotypes (Slawin et al., 2000), and provided
evidence that wild type KLK2 was a useful gene for detecting metastasis and lymph
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node involvement. Based on this method of detecting circulating cancer cells,
quantitation of KLK4 mRNA transcripts may become a useful means of detecting
disseminated cells and as a prognostic marker.
Based on the finding of elevated levels of KLK4 transcript in prostate cancer tissues, it
would also be of interest to examine the protein expression levels of hK4 in benign and
malignant prostates.
Immunohistochemical analysis of tissue sections within this
laboratory have demonstrated that hK4 is expressed to a greater degree in prostate
cancer in comparison to BPH sections (Ms L Bui, personal communication, QUT, 2003)
confirming the results documented here at the mRNA level. It may prove useful to
design specific antibodies to both the full length and the exon 1 deleted protein (and
other transcripts, if useful) in order to make a comparison between the two with respect
to protein levels in tissue extracts and blood, urine or ejaculate. Indeed, considering the
mRNA data of this study and the parallel findings with immunohistochemistry by Ms
Loan Bui, it may be that KLK4/hK4 may emerge as yet another useful kallikrein
biomarker in prostate disease.
In order to gain further information as to whether KLK4/hK4 may be expressed
temporally or differentially with progression of prostate disease, the expression profile
of KLK4 and its variants was examined in two non-malignant epithelial prostate cell
lines (RWPE1 and RWPE2), three well established prostate cancer cell lines,
representing androgen dependent prostate tumours (LNCaP) and androgen independent
tumours (DU145 and PC-3), and an in vitro metastatic cell line model (the C4 series),
derived from the prostate cancer cell line LNCaP.
Perhaps the most important finding of this part of the thesis was that full length KLK4 is
expressed only in LNCaP cells. Therefore, the LNCaP cell line was chosen as a good
model to assess KLK4 expression in subsequent hormone and growth factor regulation
experiments.
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7.2 Regulation of KLK4 Transcripts by Hormones and Growth Factors
It is well known that prostate cancer is a hormone dependent disease and consequently
many genes important to the development and progression of prostate cancer are
regulated by those same hormones and growth factors which are involved in both normal
prostate development and prostate tumourigenesis. Therefore, the rationale behind this
part of the project was to determine if KLK4 may be regulated by the hormones/growth
factors which are known to be key regulators of other genes important in cancer.
This study found that KLK4 transcript expression is increased in response to DHT, DHT
in combination with T3, and by EGF. However, in response to T3 only, no significant
changes in transcript levels were detected. As it has been established that the addition of
T3 does not cause an increase in androgen receptor levels (Zhang et al., 1999), further
experimentation is required to elucidate the mechanism by which T3 is potentially
causing an added increase in KLK4 in addition to that observed with DHT alone.
Increased expression of EGF and the EGF receptor has been linked to prostate cancer
development as evidenced by raised protein levels of EGF and the EGF receptor in
prostate cancers in comparison with benign tissue (Harper et al., 1993, Glynne-Jones et
al., 1996, Olapade-Olaopa et al., 2000). Furthermore, their expression has been
associated with prostate cancer cells undergoing progression to a more androgen
unresponsive phenotype (Schuurmans et al., 1989, Chung et al., 1992). Considering
this, it is possible that KLK4, since it is regulated by increasing levels of EGF, may also
participate in the progression to androgen independence. However, this process is very
complex and cannot be attributed to any one gene, growth factor or receptor. Therefore,
it is more than likely KLK4 may exist as one of many genes in a cascade of events
resulting in the androgen independent phenotype. In order to explore this further, it
would be interesting to examine the expression levels of KLK4 in response to EGF
treatment in the C4 LNCaP sublines which are a series of cell lines that become
increasingly metastatic and androgen insensitive. In addition, it would also be useful to
examine the response of KLK4/hK4 to EGF over a time course from 2 - 24 h,
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particularly considering MMP-7 showed the greatest response to EGF at a 24 h time
point (Sundareshan et al., 1999). Additionally, examining the expression levels of
KLK4 over a shorter time period would give some indication as to whether KLK4 is
regulated by EGF at the transcriptional level.
Many other growth factors have also been implicated in the progression of prostate
disease. These include the IGF family, TGF-α, FGF, and endothelial growth factors
which are the main stimulatory regulators of proliferation in the prostate, in addition to
the TGF-β family which is the main inhibitory regulator. Clearly, it would be interesting
to examine the effect of all of these growth factors on the expression levels of KLK4,
both at the mRNA and the protein level. While it is not possible to design primers to
detect each variant in isolation, it would be useful to perform regulation experiments to
detect the expression levels of full length KLK4 in response to various growth factors
and hormones since this transcript can be distinguished from the variants using RT-PCR.
In order to gain a better understanding of the role of KLK4 in both the androgen
dependent and androgen independent phases, conducting regulatory experiments in
androgen independent cell lines (DU145 and PC-3) would also be useful. Furthermore,
it would be interesting to examine whether KLK4 expression is induced in the RWPE1
cell line with the addition of various growth factors/hormones alone or in combination.
Complex interactions exist between growth factors/hormones and proliferative control of
cells of the prostate. Therefore, understanding the growth factor pathways as prostate
cancer progresses may lead to targeted therapy for patients with advanced disease
(Konety and Getzenberg, 1997).
Other growth factors of particular interest, considering the results obtained with the hK4
over-expressing cells as discussed below, are those associated with EMT and bone
metastasis.
Many of these are produced by osteoblasts and are incorporated into the
bone matrix or are present in the bone microenvironment. These factors include the
bone morphogenic proteins, IGF-I and IGF-II, interleukin-6, interleukin-1β, tumour
necrosis factor α (TNFα) and TGFβ (Baylink et al., 1993, Dodds et al., 1994). TGFβ
has also been linked to the process of EMT, which further highlights its role in advanced
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prostate cancer (Hay, 1995, Moustakas et al., 2002). It would be useful to examine the
effect each of these growth factors has on KLK4/hK4 expression as the association of
hK4 with EMT and bone metastases becomes clearer.
7.3 Functional Effects of hK4 Over-Expression
As KLK4 is one of the newly described members of the human kallikrein family, few
studies have focused on its potential functional role in prostate cancer. Of the research
that has been performed, a number of roles have been suggested based on sequence
similarities to other genes and biochemical studies using recombinant hK4. Due to the
similarity of hK4 to porcine enamel matrix serine proteinase 1, which is involved in the
degradation of organic matrix surrounding tooth enamel (Scully et al., 1998; Hu et al.,
2000a; Hu et al., 2000b), it has been proposed that if the substantial sequence homology
between these two enzymes parallels function, then the potential exists for hK4 to be
involved in prostate cancer metastasis to bone, the primary site for secondary tumours.
Furthermore, studies using recombinant hK4 have revealed a potential role for this
enzyme in the processes of migration and invasion due to its ability to activate pro-PSA
and single chain urokinase-type plasminogen activator (scuPA, pro-uPA) (Takayama et
al., 2001b). Prostate cancer cells over-express uPA (Van Veldhuizen et al., 1996,
Achbarou et al., 1994), which activates plasminogen to generate plasmin (Lijnen et al.,
1986), which in turn activates metalloproteases (He et al., 1989, Stricklin et al., 1977)
allowing them to digest ECM proteins, and enabling migratory cells to escape from the
primary tumour site.
These early studies have highlighted KLK4 as an important
candidate in the complex processes associated with prostate cancer progression and
metastasis. However, in order to more clearly identify these potential roles, KLK4 was
introduced into the prostate cancer PC-3 cell line and its effect on in vitro indicators of
cancer progression was studied.
No previous studies have examined the function of hK4 in an in vitro over-expression
system. Thus, perhaps the most significant outcome of this thesis was the generation of
hK4 over-expressing cells. Using these cells, a number of noteworthy observations were
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made.
The hK4 over-expressing clones have increased motility/migration across a
synthetic membrane barrier when compared with the control clones in response to a
range of different chemo-attractants from various cellular origins.
The increased
migration was partially blocked by the serine protease inhibitor, aprotinin, indicating
that the migration observed was, at least in part, due to active hK4.
This study also
demonstrated that hK4 over-expressing cells displayed increased adhesion to collagen
type I and IV, but not to fibronectin. Many of the hK4 clones also displayed altered
morphology and phenotype in comparison with the native and vector only cells, with an
increased number of processes and an irregular appearance. Furthermore, the hK4
clones also demonstrated compromised attachment to the growth surface, whereas the
native and vector only cells did not exhibit these characteristics. The evidence presented
in this thesis regarding the altered phenotype, the increased migratory potential of the
cells and the enhanced attachment to certain ECM molecules supports the theory that
hK4 could be involved in prostate tumour progression.
While this study assessed a range of hK4 clones that expressed a high, medium and low
level of KLK4/hK4, it is possible that the use of different vectors or transfection systems
may have yielded varying results. Generating stable mammalian cell lines with the
method used in this study can result in significant clonal variation due to integration of
the transfected expression vectors at random sites in the genome. This "position effect"
also compromises direct comparison of different expression constructs.
Various
superior transfection systems are now available and provide appropriate internal
controls.
These include an inducible system such as the Ecdysone-inducible
(Invitrogen), the Tet (Clontech) and the Flp-in vector system (Invitrogen).
The Ecdysone-Inducible Mammalian Expression System is designed to allow regulated
expression of the gene of interest in mammalian cells and is distinguished by its tightly
regulated mechanism that allows almost no detectable basal expression and greater than
200-fold inducibility in mammalian cells. Additionally, this system does not exert
pleiotropic effects (ie the tendency to affect the expression of multiple genes other than
the target gene) (No et al., 1996). Although this system was available within the
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laboratory, it was not attempted, as other members of the group had been unsuccessful
on several occasions with this transfection method, possibly due to cytotoxicity effects
that killed nearly all cells after addition of the two antibiotics. The Tet System provides
dose-dependent regulation whereby the level of doxycycline can be adjusted to increase
or decrease expression. Induction is reproducible so a given level of inducer provides the
same level of expression every time. In addition, the Tet system also does not exert
pleiotropic effects. Invitrogen's Flp-In System also eliminates clonal variation as it
allows direct high-level expression vectors to integrate at the same unique locus in every
transfected cell.
Generating transfected cell lines with either one of these more
advanced transfection methods would save time as they eliminate clonal variation, and
hence the need to assess a range of clones.
Although the hK4 clones that expressed a medium to high amount of hK4 performed
consistently in the functional assays, it is still uncertain whether the hK4 protein
expressed by the PC-3 cells is enzymatically active. It is possible that these cells
express the putative metalloprotease suggested to cleave hK4 at the activation site to
produce active hK4 (Takayama et al., 2001), but this has not been determined.
Alternatively, other enzymes may be present in PC-3 cells that activate pro-hK4 or it
may be auto-activated (Nicole Willemsen, personal communication, QUT, 2003).
Nevertheless, 75% of the hK4 clones’ migratory ability was blocked by the inhibitor,
aprotinin, compatible with the suggestion that active hK4 was causing migration of these
cells. Although aprotinin is a general serine protease inhibitor, and it is likely that
blocking of other serine proteases expressed by the PC-3 cells also occurred, previous
studies have shown that active recombinant hK4 does in fact bind to aprotinin
(Takayama et al., 2001), which supports the likelihood that active hK4 was blocked by
this inhibitor. Notwithstanding this, it is necessary to definitively determine whether the
enzyme is active or not.
Chromogenic assays are available, although a non-specific trypsin substrate would need
to be used since a specific hK4 substrate is not available commercially. A further
difficulty which arises when assessing enzyme activity with chromogenic assays is the
fact that the media from the hK4 over-expressing cells is likely to contain contaminating
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proteases secreted by the PC-3 cells which may activate the trypsin substrate. Therefore
a chromogenic assay may not provide an accurate reflection of the enzymatic activity of
the secreted hK4. Additionally, as has been found in other studies, it is possible that
there may not be sufficient activity of the hK4 enzyme in the secreted media. Previous
studies in this laboratory and others have found that the conditioned media from PSA
transfected cells does not demonstrate significant hydrolysis of the PSA substrate in
enzyme activity assays (T. Veveris-Lowe, QUT, personal communication, 2003,
Denmeade et al., 2003).
A method which may be more useful is to examine the proteolytic activity of the
secreted hK4 using zymography. The basis of this technique is the use of an SDS gel
impregnated with a protein or peptide substrate which is degraded by the proteases
resolved during the incubation period. Coomassie blue staining of the gel reveals sites of
proteolysis as white bands on a dark blue background. Within a certain range the band
intensity can be related linearly to the amount of protease loaded (Zhao and Russell,
2003, Kleiner and Stetler-Stevenson, 1994). Zymography using fluorogenic substrates
with a trypsin-like specificity (Zhao and Russell, 2003) could also be used to assess the
enzymatic activity of hK4, since hK4 has been shown to have trypsin-type substrate
specificity (Zhao and Russell, 2003, Nelson et al., 1999, Takayama et al., 2001).
Despite the question of whether the hK4 is enzymatically active, the hK4 overexpressing clones displayed increased motility/migration over various time points and in
response to a range of different chemo-attractants. Interestingly, the hK4 clones were
able to migrate towards soluble factors from Saos-2 cells in keeping with other studies
which suggest that prostate cancer cells preferentially disseminate to bone (Hujanen and
Terranova, 1985, Jacob et al., 1999, Bubendorf et al., 2000, Rubin et al., 2000b). It has
been demonstrated that bone extracts are potent chemoattractants over extracts from
other tissues and that they increase migration and invasion by prostate cancer cell lines
(Jacob et al., 1999). Many of these soluble factors, which include TGFβ, bFGF, KGF,
IGFs and bone morphogenic proteins, also stimulate the growth of metastatic cancer
cells in the marrow (Hauschka et al., 1986). In keeping with the potential for hK4 to
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assist in the preferential dissemination of prostate cancer cells to bone, hK4 clones
showed increased attachment to collagen IV and to collagen I, the primary matrix
molecule of bone.
Many of the hK4 clones also displayed altered morphology and phenotype in
comparison with the native and vector only cells, with an increased number of processes,
an irregular appearance in addition to compromised attachment to the growth surface.
As EMT is associated with each of these characteristics, this should be confirmed by
analysing structural targets for EMT signals, such as the re-organisation of
microfilament and actin-based cytoskeletal elements implicated in cell ruffling and
development of filopodia and lamellipodia (Savanger, 2001). A number of transcription
factors (slug/snail transcription factors; Savanger, 2001) and signaling molecules such as
the Src tyrosine kinase family and Ras family (reviewed in Boyer et al., 2000) and
TGFβ (Grande et al., 2002, Masszi et al., 2003) have been linked with cell motility and
may induce EMT (Hynes, 1992, Giancotti and Ruoslahti, 1999), along with the
activation of the integrins by extracellular signals which could also be assessed. Also
associated with EMT is the process known as ‘cadherin switching’. This occurs where
there is a change in cadherin expression in epithelial cells to a pattern similar to cadherin
expression in stromal cells (Cavallaro et al., 2002).
Specifically, N-cadherin and
cadherin-11, two mesenchymal cadherins, are up-regulated in high grade cancers and
prostate cancer cell lines that lack a functional E-cadherin-catenin adhesion complex
(Tran et al., 1999, Tomita et al., 2000, Bussemakers et al., 2000). Therefore in order to
determine whether the hK4 cells have undergone epithelial to mesenchmyal transition, it
would be useful to determine whether E-cadherin is lost from the hK4 over-expressing
cells in addition to investigating the expression and localisation of the key proteins in Ecadherin’s cellular adhesion complex. A loss of E-cadherin expression would indicate
that EMT has taken place, and therefore, the expression of the mesenchymal related
cellular adhesion molecules, N-cadherin and cadherin-11 would need to be analysed,
along with structural proteins such as vimentin and β1-containing integrins, given the
morphological changes of the cells.
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While the results obtained in this study have important implications, several other
functional assays were not performed in this study, due to time constraints.
For
example, other versions of the migration assay may also be worthwhile, such as the
‘scratch wound’ assay (Leavesley et al., 1999), where the motility of each cell line can
be characterised by assessing the ability of each cell type to move across a denuded
growth area. Clonogenic assays could be performed to evaluate the colony forming
ability of the cells. This may assist in the assessment of the tumourigenicity of the cell
types and provide evidence that hK4 expressing cells have a more metastatic phenotype.
In addition, although several adhesion assays have been performed, other extracellular
matrix (ECM) molecules should be analysed, such as vitronectin and laminin.
Finally, in vivo experimentation on the clones needs to be performed in order to assess
their tumourigenicity in nude mice, and to confirm the in vitro findings. This may be
achieved using nude mice by injecting the tranfected PC-3 cells under the kidney
capsule, a highly vascularised and suitable location for the in vivo analysis of
tumourigenicity, or by orthotopic implantation directly into the mouse prostate
(Rembrink et al., 1997).
It is possible that due to the propensity of the hK4 over-
expressing cells to migrate towards soluble factors from the Saos-2 bone cell line and
their ability to attach to collagen I, the principal component of the bone interstitial
matrix, the hK4 over-expressing cells may preferentially develop bone metastases
compared with the native PC-3 and vector only transfected cells in an in vivo model. A
number of models have been developed to assess the metastatic potential of prostate
cancer cells to bone. Tail-vein, intra-cardiac and orthotopic injection of cells have been
used quite extensively, although they result in a low frequency of osseous metastasis
formation, are complex procedures or do not sufficiently reflect the human disease
(Shevrin et al., 1988, Thalmann et al., 1994, Wu et al., 1998, Rembrink et al., 1997, An
et al., 1998). Recently a reliable and reproducible model of prostate cancer growth and
invasion in bone has been established by using intratibial inoculation with the human
prostate cancer lines PC-3 and DU145 in nude mice (Fisher et al., 2002). This model
would be of particular use to examine the interaction between the hK4 over-expressing
cells and cells of the bone environment as this method results in a high incidence of
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Chapter 7
skeletal tumour formation that is radiologically and histologically similar to those
encountered clinically.
Despite the extensive use of xenograft models to assess prostate cancer’s predilection to
develop bony metastases, these models rely on the fact that the human cells injected
must metastasise and grow in mouse organs.
It has been suggested that many of the
molecules involved in the metastatic process such as proteases, adhesion molecules,
chemotactic factors, and growth factors and their receptors, are species-specific (Nemeth
et al., 1999), which may account for the lack of osseous metastases found in some
xenograft models. To overcome this difficulty, an in vivo model of human prostate
cancer metastasis to human bone in severe combined immunodeficient (SCID) mice has
been developed (Nemeth et al., 1999). This procedure provides a useful system to study
species-specific mechanisms involved in the growth of human prostate cancer cells in
bone and the propensity of human prostate cancer cells to seed in human bone. This
method involves using macroscopic fragments of human fetal bone implanted subcutaneously into male CB.17 SCID mice. Following a four week period, human prostate
cancer cells can be injected either intravenously via the tail vein or directly into the
implanted bone chip fragment transdermally.
After six weeks, tumour growth is
assessed by palpation and magnetic resonance imaging. Studies using this model have
demonstrated that circulating human prostate cancer cells were able to specifically and
preferentially colonise implanted human bone tissue in SCID mice, and that human bone
provided a more favourable growth environment for human prostate cancer cells than
either human lung or mouse bone tissue (Nemeth et al., 1999). These observations
support the theory that the colonisation of human bone involves species- and tissuespecific mechanisms and is not due to the passive lodging of tumour cells in the bone.
This method would be useful to study the effect of the hK4 over-expressing cells’
potential involvement in the metastasis of prostate cancer cell to bone, particularly in
view of the hK4 over-expressing cells’ preferential migration towards soluble factors
from a bone cell line and enhanced attachment to collagen I.
173
Chapter 7
In vivo experiments utilising models that closely mimic human prostate cancer, such as
the intratibial inoculation method (Fisher et al., 2002) and the metastasis to human bone
in SCID mice model (Nemeth et al., 1999), may provide more valuable information to
help clarify the potential role(s) of hK4 in prostate cancer bone metastases.
7.4 Conclusion
In summary, this study has provided preliminary evidence that KLK4, as measured by
“total” KLK4 mRNA transcripts, may potentially be a useful biomarker of prostate
disease; which confirms previous observations at the immunohistochemical level.
Additionally, it has been demonstrated that KLK4/hK4 is up-regulated by the growth
factors/hormones DHT, T3 and EGF in the androgen responsive cell line, LNCaP.
The data presented here also provide new evidence which adds to our understanding of
the functional impact of hK4 over-expression in prostate cancer progression. Although
further in vitro and in vivo experimentation using these newly developed cell lines is
required to confirm the findings and determine the extent of enzymatic activity, the
results arising from this study have implicated hK4 in prostate cancer progression. This
potentially occurs via the enhanced motility displayed by the hK4 over-expressing cells
towards soluble factors from prostate and bone cells and increased attachment to
collagen I and IV. The morphological changes displayed by the over-expressing hK4
cells, possibly reflect a more metastatic phenotype, although the underlying mechanisms
of the morphological changes are yet to be established.
In conclusion, this thesis has provided evidence supporting hK4 as a molecule which
may contribute to prostate cancer progression. Additionally the elevated level of “total”
KLK4 mRNA transcripts in prostate cancer over benign disease indicates a potential role
as a biomarker for prostate disease
174
CHAPTER EIGHT
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