Atlanta University CenterDigitalCommons@Robert W. Woodruff Library, AtlantaUniversity CenterElectronic Theses & Dissertations Collection forAtlanta University & Clark Atlanta University Clark Atlanta University
Spring 5-22-2017
Activator Protein-1 in Transforming GrowthFactor-Beta Effects on Prostate Cancer CellProliferation, Migration, and InvasionCachetne S.X. BarrettClark Atlanta University, [email protected]
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Recommended CitationBarrett, Cachetne S.X., "Activator Protein-1 in Transforming Growth Factor-Beta Effects on Prostate Cancer Cell Proliferation,Migration, and Invasion" (2017). Electronic Theses & Dissertations Collection for Atlanta University & Clark Atlanta University. 61.http://digitalcommons.auctr.edu/cauetds/61
i
ABSTRACT
BIOLOGICAL SCIENCES
BARRETT, CACHÉTNE S. X. B.S. CLARK ATLANTA UNIVERSITY,
2008
ACTIVATOR PROTEIN-1 IN TRANSFORMING GROWTH FACTOR-BETA
EFFECTS ON PROSTATE CANCER CELL PROLIFERATION, MIGRATION, AND
INVASION
Committee Chair: Shafiq Khan, Ph.D.
Dissertation dated May 2017
Activator Protein-1(AP-1) family plays a central role in the transcriptional
regulation of many genes that are associated with cell proliferation, migration, metastasis,
and survival. Transforming growth factor beta (TGF-β) is a multifunctional regulatory
cytokine that regulates many aspects of cellular function, including cellular proliferation,
migration, and survival. This study investigated the role of FOS proteins in TGF-β
signaling in prostate cancer cell proliferation, migration, and invasion. DU145 and PC3
prostate cancer cells were exposed to TGF-β1 at varying time and dosage, RT-PCR,
western blot and immunofluorescence analyses were used to determine TGF-β1 effect on
FOS mRNA and protein expression levels as well as FosB subcellular localization.
Transient silencing of FOS protein was used to determine their role in cell proliferation,
migration and invasion. Our data showed that FOS mRNA and proteins were
ii
differentially expressed in human prostate epithelial (RWPE-1) and prostate cancer cell
lines (LNCaP, DU145, and PC3). TGF-β1 induced the expression of FosB at both the
mRNA and protein levels in DU145 and PC3 cells, whereas cFos and Fra1 were
unaffected and Fra2 protein expression increased in PC3 cell only. Immunofluorescence
analysis showed an increase in the accumulation of FosB protein in the nucleus of PC3
cells after treatment with exogenous TGF-β1. Selective knockdown of endogenous FosB
by specific siRNA did not have any effect on cell proliferation in PC3 and DU145 cells.
However, basal and TGF-β1-and EGF- induced cell migration was significantly reduced
in DU145 and PC3 cells lacking endogenous FosB. TGF-β1- and EGF-induced cell
invasion were also significantly decreased after FosB knockdown in PC3 cells. Transient
silencing of Fra2 resulted in decrease in cell proliferation in PC3 cells whereas transient
silencing of cFos resulted in an increase in cell number in PC3 cells. And lastly, TGF-β1
reduced FosB: cJun dimerization; cJun knockdown increased cell migration in PC3 cells
and its overexpression decreased cell migration in DU145 cells. Our data suggest that
FosB is required for migration and invasion in prostate cancer cells. We also conclude
that TGF-β1 effect on prostate cancer cell migration and invasion may be mediated
through the induction of FosB
ACTIVATOR PROTEIN-1 IN TRANSFORMING GROWTH FACTOR-BETA
EFFECTS ON PROSTATE CANCER CELL PROLIFERATION, MIGRATION, AND
INVASION
A DISSERTATION
SUBMITTED TO THE FACULTY OF CLARK ATLANTA UNIVERSITY
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR
THE DEGREE OF DOCTOR OF PHILOSOPHY
BY
CACHÉTNE SAMOIS X. BARRETT
DEPARTMENT OF BIOLOGICAL SCIENCES
ATLANTA, GEORGIA
MAY 2017
© 2017
CACHETNE SAMOIS XANAUE BARRETT
All Rights Reserve
iii
ACKNOWLEDGEMENTS
I would like to express my special appreciation to my dissertation advisor, Dr.
Shafiq A. Khan, for encouraging my research and for allowing me to grow as a research
scientist. I would also like to thank my Dissertation Advisory Committee for serving as
my committee members and for their brilliant comments and suggestions. They are: Drs.
Jaideep Chaudhary, Valerie Odero-Marah, Xiu-Ren Bu, and Adegboyega Oyelere. I
would like to thank current and former members of Dr. Khan’s lab: Cecille Ana Millena,
Dr. Silvia Caggia, Dr. Bethtrice Elliott, Clement J. Bolton II, Mawiyah Kimbrough-
Allah, and Smrruthi Venugopal. My sincerest gratitude goes to my aunt and uncle, Alma
and Del Forbes; and my friends, Trishanna Harris, MarTia Adams, Jacole Todd, and
Nikita King. I would like to especially thank my mother, Jacinth Barrett; my sisters,
Marsha Minott, Tamequa Minott, and Tahjétne Clark; my brother, Jomo Minott; my
nieces and nephews; and my Elizabeth Baptist Church Family. Words cannot express
how grateful I am for all of the sacrifices made on my behalf. Their constant prayers for
me sustained me thus far and inspired me to strive towards my goal. Finally, I would like
to express my heartfelt appreciation to my beloved Heavenly Father, whose guidance,
provision, and protection brought me here. Thanks be to God. This study was supported
by grants from NIH/NIMHD/RCMI grant #G12MD007590 and NIH/NIMHD
#5P20MD002285.
iv
TABLE OF CONTENTS
ACKNOWLEDGEMENTS………………………………………………………………iii
LIST OF FIGURES……………………………………………………………………...vii
LIST OF ABBREVIATIONS ………………………………….………………………ix
CHAPTER
I. INTRODUCTION ............................................................................................ 1
Background and Significance ........................................................................... 1
Rationale ......................................................................................................... 13
Research Question .......................................................................................... 14
Hypothesis/Specific Aims ............................................................................... 15
II. LITERATURE REVIEW ............................................................................... 21
Prostate and Prostate Cancer ........................................................................... 21
Transforming Growth Factor-Beta ................................................................. 27
Transforming Growth Factor-Beta in Prostate Development and Function ... 31
Activator Protein-1.......................................................................................... 35
Activator Protein-1 in Cancers and Prostate Cancer …………………..…….41
Activator Protein-1 in TGF-β Signaling ……………..…………………...…42
AP-1 Proteins in TGF-β Signaling and Prostate Cancer………….…….……43
III. MATERIALS AND METHODS .................................................................... 44
Chemicals and Reagents ................................................................................. 44
Human Prostate Cancer Cell Lines ................................................................. 44
Expression of FOS Family Members ...............................................................44
v
CHAPTER
RNA Isolation, cDNA Synthesis, and RT-PCR.............................................. 45
Western Blot Analyses ……………………………………………………....46
Immunoflurescence of FosB ………………………………………………...47
Transfections ……………………………………………………………...…48
Cell Proliferation Assays …………………………………………………....50
Cell Migration Assays ……………………………………………………….51
Cell Invasion Assays ………………………………………………………...51
Co-immunoprecipitation …………………………………………………….52
Statistical Analyses ………………………………………………………….53
IV. RESULTS ....................................................................................................... 54
Expression of FOS Family Members in Prostate Cancer Cells ..................... 54
TGF-β Effects on FOS protein Expression and Nuclear Accumulation ......... 56
FosB Knockdown on TGF-β1 mediated cell prolifaretion, migration ............ 58
And Invasion
FOS Family Members Role in Prostate Cancer Cell Growth ……………….61
and Poliferation
TGF-β1 Effect on AP-1 dimerization ……………………………………….65
The Role of cJun Protein in Prostate Cancer Cell Migration…..……………66
V. DISCUSSION………………………………………………………………..69
VI. CONCLUSION………………………………………………………………74
APPENDIX
A. Table 1……………………………………………………………………….76
vi
REFERENCES ………………………………………………………………………….77
vii
LIST OF FIGURES
Figure
1. TGF-β role in cancer ................................................................................................ .3
2. Schematic of TGF-β signaling ................................................................................ 5
3. Prostate anatomy and prostate cancer risk factors ................................................. .21
4. Leading sites of new cancer cases and deaths-2016 estimates ............................... 23
5. Stages of prostate cancer ....................................................................................... 25
6. Transformed metastatic prostate cells ................................................................... 26
7. Schematic representation of TGF-β-receptors ...................................................... 29
8. TGF-β1 in prostate cancer progression ................................................................. .33
9. Schematic diagram showing JUN and FOS molecular structure ......................... 38
10. FOS family basal expression ................................................................................ 55
11. TGF-β1 on FOS Family mRNA and protein expression ....................................... 57
12. The effect of FosB knockdown on TGF-β1-induced prostate cancer cell
Proliferation ........................................................................................................... 59
13. The effect of FosB knockdown on TGF-β1-induced prostate cancer cell
Migration............................................................................................................... 60
14. Effects of FosB knockdown on prostate cancer cell invasion .............................. 61
15. The effect of cFos and Fra1 knockdown on prostate cancer cell number ........... 63
16. Effects of Fra2 knockdown on prostate cancer cell proliferation ........................ 64
viii
17. FOS family knock down on prostate cancer cell morphology .............................. 65
18. The effect of TGF-β1 on FosB: cJun dimerization ................................................ 66
19. The effect of cJun knockdown on prostate cancer cell migration ....................... 67
20. The effect of cJun overexpression on prostate cancer cell migration ................... 68
ix
LIST OF ABBREVIATIONS
AKT Protein Kinase B
AMH Anti-Müllerian Hormone
ANOVA Analysis of Variance
AP-1 Activator Protein 1
ATF Activating Transcription Factor
bp base pair
BMPs Bone Morphogenetic Proteins
BSA Bovine Serum Albumin
bZIP Basic Leucine Zipper Domain
CDC42 Cell Division Control Protein 42
cDNA Complementary Deoxyribonucleic Acid
CSCs Cancer Stem Cells
DAPI 4’-6-Diamidino-2-phenylindole
DBD DNA Binding Domain
DNA Deoxyribonucleic Acid
dNTP Dinucleotide Triphosphate
ECL Enhanced Chemiluminescence
EGF Epidermal Growth Factor
ERK1/2 Extra-cellular Signal Regulated Kinases
FBS Fetal Bovine Serum
x
Fra1 Fos-Like Antigen 1
Fra2 Fos-Like Antigen 2
FOS Finkel-Biskis-Jinkins murine osteogenic sarcoma virus
GDF Growth and Differentiation Factor
GTPase Guanosine Triphosphate Hydrolytic Enzymes
HIPK2 Homeodomain Interacting Protein Kinase 2
IE Immediate Early
IgG-HRP Immunoglobulin Horseradish Peroxidase
JNK JUN N-Terminal Kinases
KSFM Keratinocyte Serum Free Medium
LAP Latency-associated Peptide
LZD Leucine Zipper Domain
MAPK Mitogen-activated Protein Kinases
MEM Minimum Essential Medium
MIS Müllerian Inhibiting Substance
MMP1 Matrix Metalloproteinase
mRNA Messenger Ribonucleic Acid
MTS 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl) -2-(4-
sulfophenyl)-
2H-tetrazolium inner salts
MTT 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
mTOR Mechanistic Target of Rapamycin
NCBI National Center for Bioinformatics
NF-κB Nuclear Factor κB
xi
NIH National Institutions of Health
NLM National Library of Medicine
PAI1 Plasminogen Activator Inhibitor 1
PBS Phosphate Buffered Saline
PCa Prostate Cancer
PCR Polymerase Chain Reaction
PI3K Phosphoinositide-3-Kinases
PSA Prostate Specific Antigen
PTEN Phosphatase and Tensin Homolog
PVDF Polyvinylidene Difluoride
RAC1 Ras-Related C3 Botulinum Substrate 1
RT Reverse Transcriptase
Rh recombinant human
Rho Rhodopsin
RNA Ribonucleic Acid
RPMI Roswell Park Memorial Institute
SARA Smad Anchor for Receptor Activation
SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
Smad Mothers against Decapentaplegic
SOX4 SRY-Related HMG-Box 4
TAD Transactivation Domain
TβRI Transforming Growth Factor-β Type I Receptor
TβRII Transforming Growth Factor-β Type II Receptor
xii
TGF-β Transforming Growth Factor-β
TPA 12-O-Tetradecanoyl-phorbol-13-acetate
TRE TPA-Response Element
UV Ultraviolet
1
CHAPTER 1
INTRODUCTION
Background and Significance
Cancer is a key health issue across the world, causing substantial patient
morbidity and mortality.1 Prostate cancer (PCa.) is the most common malignant disease in
males and the second leading cause of cancer related deaths in men in developed
countries.2-3 Prostate cancer accounts for 28% of cancer diagnoses and 10% of cancer
deaths in men.3 Patients with localized prostate cancer have a relatively long-term
survival due to great advances in surgical resection and adjuvant therapy.4 However,
patients with advanced, especially metastatic bone disease are often associated with a
poor prognosis.4 Studies have shown that around 30% of these patients will develop
distant metastases within five years of diagnosis, even after radical surgery.4 Bone
metastases occur in more than 80% of cases of advanced-stage prostate cancer, which
confers a high level of morbidity, with a five-year survival rate of 25% and a median
survival of approximately 40 months.4 Patient prognosis is tightly linked with metastatic
dissemination of the disease to distant sites, with metastatic diseases accounting for a vast
percentage of cancer patient mortality.1 A critical barrier for the successful prevention
and treatment of recurrent prostate cancer is detection and
2
eradication of metastatic and therapy-resistant disease.5 While advances in this area have
been made, the process of cancer metastasis and the factors governing cancer spread and
establishment at secondary locations are still poorly understood.6
With rare exceptions, the natural history of all types of tumors is known to
progress from localized indolent stages to aggressive metastatic stages.7-9 Recent
advancements in biomarker research have made significant progresses to help prediction
of cancer progression and disease outcome.9-12 However, the molecular mechanisms
behind tumor progression remain elusive. Transforming growth factor beta (TGF-β) is
known to inhibit cell cycle in benign cells but promote progression and metastasis in
cancer cells (Figure 1),9, 13-14 a phenomenon known as TGF-β paradox.9, 15 Although there
are numerous articles with different approaches tackling this topic, to date, a logical
explanation leading to TGF-β paradox remains elusive and is accepted as a scientific
mystery.9, 14-17
3
Figure 1: The Role of TGF-β in Cancer; In normal and premalignant cells, TGFβ
enforces homeostasis and suppresses tumor progression directly through cell-autonomous
tumor-suppressive effects (cytostasis, differentiation, apoptosis) or indirectly through
effects on the stroma (suppression of inflammation and stroma-derived mitogens).
However, when cancer cells lose TGFβ tumor-suppressive responses, they can use TGFβ
to their advantage to initiate immune evasion, growth factor production, differentiation
into an invasive phenotype, and metastatic dissemination or to establish and expand
metastatic colonies.
The TGF-β superfamily comprises TGF-β1–3, bone morphogenetic proteins
(BMPs), growth and differentiation factors (GDFs), Nodal, activins/inhibins, Müllerian
inhibiting substance (MIS)/anti-Müllerian hormone (AMH), and Lefty. These ligands
were initially grouped accordingly to the functional roles observed following their
4
original identification.18-21 As it becomes clear that most ligands play multiple functions
depending on cell type, developmental stage, or tissue conditions, they are now classified
by sequence similarity and the downstream pathway they activate.22 Each family member
has an overall basic structure, in which inactive forms are produced with an N-terminal
secretion peptide and a large pro-peptide domain known as latency-associated peptide
(LAP). Cleavage of the pro-peptide domain by pro-protein convertases releases a mature
domain at the C-terminus, which eventually dimerizes.23 The pro-peptide domain has
major regulatory roles. It influences protein stability and functions as chaperone during
secretion, also mediating diffusion through interactions with the extracellular matrix and
inhibiting the active peptide form even after cleavage.24-26 Secreted cytokines of the TGF-
β family are found in all multicellular organisms and implicated in regulating
fundamental cell behaviors such as proliferation, differentiation, migration and survival.23
Virtually, all types of cells produce and are sensitive to TGF-β superfamily members.27
TGF-β is a pleiotropic factor with several different roles in health and disease.28-29 As
stated earlier, in cancer, TGF-β plays a paradoxical role, it represses epithelial tumor
development in the early steps of tumorigenesis, while in advanced stages it can stimulate
tumor progression.29-31 In epithelial cells, TGF-β has anti-proliferative and apoptotic roles
which enable it to reverse local mitogenic stimulation in the pre-tumoral stage in the
epithelium.29 During the advance of tumorigenesis, carcinoma cells acquire resistance to
the proliferative inhibition and apoptosis induced by TGF-β. Several mechanisms have
been described to explain the changes in the response of tumor cells to TGF-β1, including
mutations in the machinery of TGF-β signaling.29
5
Mammals express three genetically distinct isoforms of TGF-𝛽 (TGF-𝛽1, TGF-
β2, and TGF-β3) with high homology. The TGF-β1, β2, and β3 genes are located on
chromosomes 19q13, 1q41, and 14q24, respectively.28-29 TGF-𝛽 initiates signaling by
binding to cell-surface serine/threonine kinase receptors types I and II (TβRI and TβRII,
respectively), which form a heteromeric complex in the presence of the dimerized ligand
(Figure 2).
Figure 2. Schematic Diagram of TGF-β Signaling from Cell Membrane to the Nucleus
The arrows indicate signal flow and are color coded: orange for ligand and receptor
activation, gray for Smad and receptor inactivation, green for Smad activation and
formation of a transcriptional complex, and blue for Smad nucleocytoplasmic shuttling.
Phosphate groups and ubiquitin are represented by green and red circles, respectively.
Binding of TGF-𝛽 to TβRII leads to the phosphorylation of TβRI, thus activating its
kinase domain.29, 32 When the receptor complex is activated, it phosphorylates and
stimulates the cytoplasmic mediators, Smad2 and Smad3.29, 33 The phosphorylation of
6
Smad2/3 releases them from the inner surface, where they are specifically retained by
Smad anchor for receptor activation (SARA). Further on, Smad2/3 forms a heterotrimeric
complex with the common Smad4, which is then translocated into the nucleus where, in
collaboration with other transcription factors, it binds and regulates promoters of different
target genes.28-29, 33 In addition to Smad signaling, TGF-𝛽1 may activate other intracellular
signaling pathways, called non-Smad pathways, such as mitogen-activated protein
kinases (MAPK): ERK1,2, JNK and p38; PI3K (phosphoinositide 3-kinase)/AKT1/2 and
mTOR, known as cell survival mediators; NF-𝜅B (nuclear factor 𝜅B), Cyclooxygenase-2,
and prostaglandins; and the small GTPase proteins Ras, Rho family (Rho, Rac1 and
Cdc42), among others.29, 34-35
TGF-β overproduction is a universal event in cancer cells and is a poor
prognostic marker.29, 36-40 The mechanism, through which TGF-β regulates its own
production, is different between benign and cancer cells. Under the normal physiological
conditions, the level of TGF-β is tightly regulated within the microenvironment through a
negative feedback loop to maintain a relatively constant level of TGF-β. Too little or too
much TGF-β will have an unfavorable consequence.29, 41-43 However, this principle does
not apply to cancer. Cancer cells, especially the advanced cases, are capable of evading
the immune surveillance program due to the well-known phenomenon of auto-induction
of TGF-β by cancer cells,29 resulting in an elevated TGF-β in the microenvironment
through a positive feedback loop.29, 44 As a result, there is an accumulation of TGF-β in the
microenvironment, which further promotes tumor progression.29, 36, 39, 45 According to the
current literature and experimental evidence, TGF-β is a potent ligand that regulates
7
carcinoma initiation, progression and metastasis through a broad and complex spectrum
of interdependent interactions.30 The knowledge about the mechanisms involved in TGF-
β signal transduction has allowed a better understanding of the disease pathogenicity as
well as the identification of several molecular targets with great potential in therapeutic
interventions.28
It is also becoming apparent that TGF-β signaling intersects with several
transcription factors and regulators, such as GL1, SOX4, Tieg3/Klf11, Id, and AP-1
proteins.46-50 Many studies have implicated Activator Protein-1 (AP-1) proteins in TGF-β
signaling.50-52 Numerous studies have characterized the differential expression of specific
genes in response to TGF-β, revealing a common link in the ability of TGF-β to regulate
many of these genes through the functions of the AP-1 family of transcription factors.53
The ability of TGF-β to induce the expression of several genes, including PAI-1,
clusterin, monocyte chemoattractant protein-1 (JEyMCP-1), type I collagen, and TGF-β1
itself depends on specific AP-1 DNA-binding sites in the promoter regions of these
genes.53-58 Furthermore, TGF-β-mediated transcriptional activation of several of these
genes requires AP-1 proteins.53-54, 56-58 Intriguingly, the expression of many AP-1 proteins
themselves is induced as an early response to TGF-β in a cell type-specific manner.53, 59-60
It has been demonstrated that this induced expression of particular AP-1 family members
is involved in TGF-β-mediated regulation of subsequent target genes.53, 58 In addition,
genetic studies of TGF-β signaling in Drosophila melanogaster reveal a direct overlap
between AP-1 and TGF-β signaling and suggest an evolutionarily conserved convergence
of these pathways.53, 61 Together, these studies demonstrate a link between TGF-β
8
signaling and AP-1 proteins in the TGF-β-regulated expression of various genes. The
molecular mechanisms responsible for the TGF-β-mediated transcriptional activation of
these genes are just beginning to be elucidated.53
The AP-1 family consists of dimeric protein complexes composed of different Jun
proteins (c-Jun, JunB, and JunD) and four FOS proteins (c-Fos, FosB, Fra1, and Fra2).
These proteins form Jun-Jun homodimers and Jun-Fos heterodimers and bind to the 12-
O-tetradecanoylphorbol-13-acetate response element, TGACTCA palindromic sequence,
in the promoters of target genes.50, 62-63 AP-1 proteins have been shown to be involved in
cell proliferation, inflammation, differentiation, apoptosis, wound healing, and
carcinogenesis.50, 64-65 The transcription factor AP-1 converts extracellular signals into
changes in the expression of specific target genes which harbor AP-1-binding site(s) in
their promoter and enhancer regions. AP-1 proteins are certainly important participants
and possibly determinant factors in the diverse mechanisms that contribute to the
development of human cancers, although casual proof for these functions is yet to be
established. The fact that AP-1 is positioned as a signal responsive transcription factor
complex at the end of a large number of signaling cascades, makes it very likely that AP-
1 components could provide the missing link between growth factor signaling and the
cell cycle machinery.
c-Fos and c-Jun have been extensively characterized and studied following their
identification as the original components of AP-l.66 Accordingly, a number of researchers
assume that what 'has been mostly worked for Jun' (and Fos) 'serves as a useful paradigm
for its other family members.66-67 However, as indicated by differences in the kinetic rates
9
of induction, modes of regulation and transactivation properties of each of these genes,
this view can only hold true to a point.66 Ultimately, each FOS and JUN gene member
should be investigated individually to gain a better understanding of the overall function
of AP-1.66 Previous studies in our laboratory have elucidated the roles of JUN proteins in
prostate cancer cell proliferation giving rise to this research project’s focus on
understanding the individual roles of FOS family members in prostate cancer cell
development and progression.
AP1 mediation of cellular response to growth factors is suggested by the
observation that deregulated expression of certain members of the Fos and Jun families
results in the neoplastic transformation of susceptible cells.68 Studies revealed that AP-1
DNA binding activity is not a single transcription factor but a dimer.69 Different AP-1
subunits display functional diversity in a cell-type specific manner 70 and different subsets
of AP-1 proteins have differing dimerization requirements.71 cJun, for example, can
homo- and heterodimerize while cFos can only form heterodimers. These AP-1 dimers
regulate a wide variety of cellular processes including the immune response, cell
proliferation, apoptosis, and tumorigenesis.63, 71 Different dimer compositions showed
promoter-specific differences in activating transcription of reporter genes.71-73 The role of
AP-1 proteins has been widely studied; however, discerning the distinct roles of
individual dimer compositions remains challenging.71 This project arose from studies
indicating that JunD plays an essential role in the proliferation of prostate cancer
epithelial cancer cells.50 It has also been suggested by several studies that expression of
AP-1 proteins is associated with a more aggressive clinical outcome in prostate cancer
10
patients.50, 74-75 Most of these studies have, however, focused on the expression and/or
function of activated AP-1 complex containing c-Jun and c-Fos.50, 76 Consequently, the
specific functions of individual AP-1 family members and various homo- and
heterodimers in the regulation of specific cellular processes remain largely unknown.50
Studies also showed that Jun-Fos dimers that have similar DNA binding specificities can
differ in transcriptional activity due to non-conserved domains located outside the bZIP
region that can be regulated by phosphorylation. It is therefore plausible that AP-1 dimers
of different composition execute specific cellular programs.73 With this in mind it is our
desire to decipher the individual roles of the FOS family members in prostate cancer cell
proliferation, migration and invasion and in the process determine which FOS family
member could be the partner for JunD and is essential for prostate cancer cell
proliferation.
cFos has been found to induce differentiation of certain cell types,66, 77 and to also
transform cells following its overexpression and removal of part of its 3' untranslated
region.66, 78 cFos may repress its own transcriptional activation and the activation of other
genes such as Egr-l,66, 79 in addition to trans-activating the expression of genes like
collagenase and Fra-1.66, 80-81 In oral cancer it has been observed that cFos/JunD
heterodimer together showed higher transcriptional activity than JunD/JunD
homodimers.73, 82 Therefore, it was speculated that JunD/JunD homodimer formation
might prevent the precancerous cells entering into cancerous condition, but as soon as
participation of cFos member takes place in AP-1 complex formation, the precancerous
cells are pushed further in the aggressive cancerous condition.82 High cFos protein levels
11
significantly correlate with high MMP9 expression and both proteins are weakly
associated with a positive nodal status in breast cancer patients.83-85 cFos expression also
correlated with cyclin E which is another indicator of unfavorable outcome in breast
cancer patients.85-86 In transient transfection experiments, cFos overexpression led to a
weak (1.65–1.81-fold) stimulation of migration and invasion through matrigel
membranes in MCF7 cells, whereas in MDA-MB231 cells, the invasive potential was
non-significantly enhanced independent of cell migration.85 These data point to a
stimulating effect of cFos on cell invasion and are in accordance with its oncogenic
function observed in various cell systems.85, 87
FosB expression is statistically associated with a well-differentiated, estrogen-
and progesterone- receptor positive phenotype of breast cancer samples, FosB expression
was associated with higher levels of the cell-cycle inhibitor Rb and low expression of the
proliferation marker Ki67.85-86 FosB expression was statistically associated with the
collagenase MMP1, which also correlates with a positive estrogen receptor status, and
surprisingly, with a nodal-negative tumor type.85 In invasion assays with breast cancer
cell lines, FosB had no significant influence on invasion of MDA-MB231 cells, whereas
in MCF7 cells, the number of invasive cells was significantly increased after transient
transfection with FosB expression vectors.85 Yet, the stimulating effect was even stronger
in control inserts without matrigel membrane indicating that in MCF7 cells, FosB
stimulates cell migration through the pores of the insert bottom, whereas the relative
number of invasive cells is not increased.85 These data suggest that in mammary
carcinomas, FosB is probably not involved in tumor invasion.85 Mice studies reveal that
12
mice lacking FosB develop normally but display a profound nurturing defect whereas,
overexpression of ΔFosB (an alternative spliced form of FosB which lacks transactivation
activity but binds JUN proteins and DNA with similar efficiency as FosB) interferes with
normal cell differentiation.88
Like other AP-1 subunits, Fra1 has been recently linked to multiple cancers,
including breast, bladder, colon and esophagus cancers and head and neck squamous cell
carcinoma.70 Fra1 is highly expressed in many epithelial cancers including squamous cell
carcinoma of the skin (cSCC) and head and neck squamous cell carcinoma (HNSCC).
However, the functional importance and the mechanisms mediating Fra1 function in
these cancers are not fully understood.70 While c-Jun was required for the expression of
the G1/S phase cell cycle promoter CDK4, Fra1 was essential for AKT activation and
AKT-dependent expression of CyclinB1, a molecule required for G2-M progression.70
Exogenous expression of a constitutively active form of AKT rescued cancer cell growth
defect caused by Fra1-loss.70 Additionally, Fra1 knockdown markedly slowed cell
adhesion and migration, and conversely expression of an active Fra1 mutant (Fra1DD)
expedited these processes in a JNK/c-Jun-dependent manner.70 Fra1 only weakly
transforms rat embryo fibroblasts and causes no overt morphological transformation of
rat embryo fibroblasts.66, 81 The weak transforming potential of Fra-1 is due to a lack of a
C-terminal transactivation domain,66 and so Fra-1 tends to repress the expression of IE
genes,66, 79 particularly in combination with JunB and c-Jun.66, 89 However, when
complexed with JunD, Fra-1 may stimulate AP-l-dependent transcription.66, 89 Fra-1
expression is strong in highly invasive cell lines like MDA-MB231, but negative or weak
13
in more differentiated breast cancer cell lines (MCF7, T47D).85 In clinical tumor tissues,
only very low Fra-1 protein levels were found by Western blot analysis.85 Prior study on
mammary carcinomas, showed Fra-1 expression was significantly associated with a
poorly differentiated, estrogen-receptor negative phenotype and strong Ki67 and Cyclin E
expression.85-86
Invasion assays showed a significant increase of the invasive potential after
forced Fra-2 overexpression in MDA-MB231, but not MCF7 cells. In clinical tumor
tissue, total Fra-2 expression was significantly associated with high Cyclin D1 and Cyclin
E expression.85-86 Fra-2 protein levels and expression of the more slowly migrating,
phosphorylated Fra-2 bands correlated with high expression of MMP9, PAI-1 (both
indicators of unfavorable outcome) and the PAI-1/uPA and PAI-1/tPA complexes.85 In
addition, those same studies showed a significantly higher frequency of recurrence in
patients with high levels of the phosphorylated Fra-2.85
Rationale
Prostate cancer (PCa) is the most diagnosed cancer and the second leading cause
of cancer death among men in the United States.90-91 TGF-β was originally described as
being one of the most potent polypeptide growth inhibitors isolated from natural
sources.92 TGF-β is a secreted cytokine that acts as a major anti-proliferative factor in the
initial stages of prostate cancer, whereas in the advanced stages of prostate cancer, it
acquires pro-oncogenic and pro-metastatic properties.50, 93-95Deregulation of TGF-β
expression or signaling has been implicated in the pathogenesis of a variety of diseases,
including cancer. There is growing evidence that in the later stages of cancer
14
development, TGF-β is actively secreted by tumor cells and does not merely act as a
bystander but rather contributes to the cell growth, invasion, and metastasis and decreases
host-tumor immune response.96-97 Activator protein-1 (AP-1) was one of the first
transcription factors to be identified, but its physiological functions are still being
unraveled. AP-1 activity is induced by a plethora of physiological stimuli and
environmental insults98 such as growth factors, cytokines, tumor-promoters and UV-
irradiation.99 In turn, AP-1 regulates a wide range of cellular processes, including cell
proliferation, death, survival and differentiation.98 Although AP-1 proteins share a high
level of sequence and function homology, they exhibit distinct expression patterns and
differ in their transcriptional and biological activities.100
Research Question
Previous studies have shown different effects of TGF-β1 on proliferation of
different prostate cancer cell lines; TGF-β inhibits proliferation of DU145 cells but has
no effect on proliferation of PC3 cells in the presence of functional TGF-β receptors and
Smad signaling,101-104 indicating differences in signaling mechanisms in two cell lines
downstream of receptor-dependent Smad activation that are responsible for differential
effects of TGF-β on cell proliferation.50 Other intracellular proteins influence TGF-β
effects,50, 105-107 and studies have shown that TGF-β signaling interacts with several
transcription factors including AP-1. In prostate cancer, expression of AP-1 proteins has
been associated with disease recurrence and more aggressive clinical outcome.50, 108-109
Previous studies have shown that without JunD prostate cancer cells do not proliferate,
those same studies indicated that JunB and cJun knockdown had no effect on cell
15
proliferation in prostate cancer cells; considering the fact that AP-1 proteins must
function as dimers, neither JunB or cJun knockdown affected prostate cancer cell
proliferation, and that JUN proteins are able to both homo and heterodimerize, the
question becomes: which AP-1 protein could potentially be a partner for JunD and is
required for prostate cancer cell proliferation to occur? In an attempt to answer this
question there are some other primary queries that must be answered. Therefore, this
project is designed to determine: 1) Are FOS family proteins expressed in prostate cancer
cell lines and is their expression being regulated by TGF-β? 2) Does the presence of
TGF-β influence FOS protein subcellular localization and dimerization? 3) Are FOS
proteins involved in TGF-β effects on prostate cancer cell proliferation, migration, and
invasion?
Hypothesis
With these questions in mind we hypothesize that JunD is essential for prostate
cancer cell proliferation; therefore, without JunD, prostate cancer tumors would not
develop. In addition, JunD does not work alone; it requires another AP-1 partner such as
a member of JUN or FOS family to exert its effects on cancer cell proliferation and tumor
development. JUN and FOS proteins share extensive homology within the leucine zipper
and basic domains. However, despite their homology, these proteins display different
transcriptional activity. Therefore, we also hypothesize that the FOS proteins contribute
distinct functions towards the activity of the AP-1 heterodimers and that TGF-β1 could
induce the expression of FOS proteins and these proteins could play an important role in
prostate cancer cell proliferation, migration, and invasion.
16
Specific Aims
In an attempt to test the above hypotheses and answer the research questions
discussed above, the following aims have been addressed:
Specific Aim 1: To determine the basal expression of FOS family members (FosB, cFos,
Fra1, and Fra2) mRNA and protein in normal prostate epithelial cells and prostate cancer
cells, and determine if their expression is regulated by TGF-β1.
Rationale: The transcription factor AP-1 is activated in response to an incredible array
of stimuli, including mitogenic growth factors, inflammatory cytokines, growth factors of
the TGF-β family, UV and ionizing irradiation, cellular stress, antigen binding, and
neoplastic transformation.110 The AP-1 transcription factor consists of a large set of dimer
combinations formed between the Jun, Fos and ATF families of proteins.111-113 AP-1
activity converts extracellular signals into changes in gene expression patterns through
the binding of AP-1 dimers to specific target sequences located within the promoters and
enhancers of target genes. These targets include genes important for regulating many
biological processes including proliferation, differentiation, apoptosis and
transformation.110, 112, 114-115 AP-1 activity functions in a hierarchy: dimerization of AP-1
proteins is required for DNA binding that in turn leads to transcriptional activity.112 TGF-
β acts as a tumor suppressor in early stages of the tumor development, and then switches
to a tumor promoter in later stages of tumor development52, 62-63 through undefined
mechanisms. Many TGF-β1 regulated genes have AP-1 binding sites53-58 and AP-1
themselves are being regulated by TGF-β1.53, 59-60 Previous studies in our lab showed that
all JUN family members are constitutively expressed in various prostate cell lines at the
17
mRNA level but exhibited differences in the levels of JUN proteins in various cell lines,
indicating differential regulation of proteins in different cell lines.50 These results also
showed that TGF-β induces a reduction in JunD protein levels in DU145 cells but not in
PC3 cells. Because JunD is required for cell proliferation, these results suggest that
reduction of JunD levels in DU145 cells in response to TGF-β may lead to reduction in
cell proliferation in these cells.50 On the other hand, the lack of TGF-β effects on PC3 cell
proliferation may be due to their resistance to TGF-β-induced reduction of JunD levels.50
Previous studies have revealed the response of JUN proteins to TGF-β1 stimulation and
even demonstrated that JunD is essential for TGF-β1-induced effects on prostate cancer
cell proliferation; however, it is still unclear whether FOS proteins are influenced by
TGF-β1 and could be essential for TGF-β effects on prostate cancer cell growth and
progression.
Experimental Design: The steady state expression of FOS mRNA and protein was
determined using prostate and prostate cancer epithelial cells seeded in 10 cm dishes and
allowed to grow for 48 hours; at which point the cells will be lysed and both RNA and
protein will be extracted and analyzed for FOS mRNA and protein expression using
reverse transcription polymer chain reaction (RT-PCR) and Western Blot respectively.
Next, DU145 and PC3 prostate cancer cells lines will be seeded to 80% confluency in 6-
welled plates and treated with 5ng/ml of exogenous TGF-β1 at varying time points (0-8
hours) as well as varying concentrations of TGF-β1 (0-10ng/ml) for four hours. The cells
will be lysed and analyzed for mRNA and protein expression using RT-PCR and Western
18
Blot analyses. This should reveal the effects of TGF-β1 on FOS mRNA and protein
expression.
Specific Aim 2: To determine the effects of FOS family members in TGF-β regulated cell
proliferation, migration and invasion of prostate cancer cells.
Rationale: AP-1 subunits bind to a common DNA site, the AP-1-binding site. As the
complexity of our knowledge of AP-1 factors has increased, our understanding of their
physiological function has decreased.114 This trend, however, is beginning to be reversed
due to the recent studies of gene-knockout mice and cell lines deficient in specific AP-1
components.114 Such studies suggest that different AP-1 factors may regulate different
target genes and thus execute distinct biological functions.114 Also, the involvement of AP-
1 factors in functions such as cell proliferation and survival has been made somewhat
clearer as a result of such studies.114 In addition, there has been considerable progress in
understanding some of the mechanisms and signaling pathways involved in the
regulation of AP-1 activity.114 AP-1 activity is regulated in a given cell by a broad range
of physiological and pathological stimuli, including cytokines, growth factors, stress
signals and infections, as well as oncogenic stimuli.62 Regulation of net AP-1 activity can
be achieved through changes in transcription of genes encoding AP-1 subunits, control of
the stability of their mRNAs, posttranslational processing and turnover of pre-existing or
newly synthesized AP-1 subunits, and specific interactions between AP-1 proteins and
other transcription factors and cofactors.62 Previous studies in our lab using transfection
of DU145 and PC3 prostate cancer cells with JunD siRNA caused a significant
reduction in proliferation of both DU145 (82% inhibition, p < 0.05) and PC3 (71%
19
inhibition, p < 0.05) cells. On the other hand, knockdown of either c-Jun or JunB had no
significant effect on cell proliferation in both cell lines. These results suggested that JunD
is required for proliferation of both DU145 and PC3 cells.50 However, the specific roles
of individual AP-1 family members in the development and progression of prostate
cancer are still largely unknown.
Experimental Design: DU145 and PC3 cells lines will be seeded in 6-welled plates and
grown to 80 % confluency; the cells will be transiently transfected to knockdown the FOS
proteins using siRNA specific for FosB, cFos, Fra1 and Fra2. The transfected cells will
be used to perform proliferation, migration and invasion assays (MTT, transwell inserts
and matrigel, respectively).
Specific Aim 3: To identify which AP-1 protein(s) dimerizes with JunD and is/are
essential for regulating cell proliferation in prostate cancer cells.
Rationale: Recent studies using cells and mice deficient in individual AP-1 proteins have
begun to shed light on their physiological functions in the control of cell proliferation,
neoplastic transformation and apoptosis.115 The main characteristic of the AP-1
complexes in the cell is their heterogeneity in dimer composition.113 This heterogeneity is
caused by the fact that multiple AP-1 sub-units can be expressed at the same time,
including c-Jun, JunB, JunD, c-Fos, FosB, Fra1, Fra2, ATF2, ATFa and ATF3.113 The
actual activities of JUN: FOS depend on the cell type, its differentiation state and the
type of stimuli it has received.113 Earlier studies have shown that dimerization is a
requirement for activation of AP-1 proteins and that AP-1 proteins form multiple homo-
and heterodimers; and the composition of these dimers may dictate expression of specific
20
genes involved in specific biological responses. However, the specific roles of individual
AP-1 family members in the development and progression of prostate cancer are still
largely unknown. Few reports have shown the effects, if any, of TGF-β on AP-1 in
prostate cancer.116-118
Experimental Design: DU145 and PC3 cells lines will be seeded in 6-welled plates and
grown to 80 % confluency; the cells will be transiently transfected to knockdown the FOS
proteins using siRNA specific for FosB, cFos, Fra1 and Fra2. The transfected cells will
be used to perform proliferation assays direct cell count, and MTT respectively.
21
CHAPTER 2
LITERATURE REVIEW
Prostate and Prostate Cancer
The prostate, an androgen-regulated exocrine gland, is an integral part of the male
reproductive system (Figure 3A) which has an essential function in sperm survival and
motility.119 It is located immediately below the bladder and just in front of the bowel. Its
main function is to produce fluid which protects and enriches sperm. In younger men, the
prostate is about the size of a walnut (Figure 3B).
22
Figure 3: The Prostate A) Anatomy of the human prostate, B) Normal prostate compared
to an enlarged prostate, C) Factors that promote prostate health, D) Factors that enhance
prostate cancer risks
It is doughnut shaped as it surrounds the beginning of the urethra (the tube that conveys
urine from the bladder to the penis). The nerves that control erections surround the
prostate.91 Prostate cancer is usually one of the slower growing cancers. In the past, it was
most frequently encountered in men over 70, and many of those men died of other causes
before their prostate cancer could kill them.120 This led to the old saying “most men die
with, not of, prostate cancer.” However, that is certainly not true today. Three
developments have changed things considerably: 1) Men are living longer, giving the
cancer more time to spread beyond the prostate, with potentially fatal consequences. 2)
More men in their early sixties, fifties and even forties are being diagnosed with prostate
cancer.120 Earlier onset combined with the greater male life expectancy means those
cancers have more time to spread and become life-threatening unless diagnosed and
treated. 3) Prostate cancer in younger men often tends to be more aggressive and hence
more life-threatening within a shorter time. Those diagnosed at a young age have a higher
cause-specific mortality than men diagnosed at an older age, except those over age 80
years.120 Early-onset prostate cancer has a strong genetic component, which indicates that
young men with prostate cancer could benefit from evaluation of genetic risk.120
Furthermore, although the majority of men with early-onset prostate cancer are diagnosed
with low-risk disease, the extended life expectancy of these patients exposes them to
long-term effects of treatment-related morbidities and to long-term risk of disease
progression leading to death from prostate cancer.120
23
Prostate cancer is the most diagnosed and the second leading cause of cancer
deaths among American men (Figure 4).
According to American Cancer Society, 180,896 men will be diagnosed and 26,120 men
will die of prostate cancer in US in 2016. Cancer statistics for 2016 indicates that apart
from skin cancer prostate cancer is the most frequently diagnosed cancer in men. For
reasons that remain unclear, the risk of prostate cancer is 70% higher in blacks than in
non-Hispanic whites. With an estimated 26,120 deaths in 2016, prostate cancer is the
second-leading cause of cancer death in men. Prostate cancer death rates have been
decreasing since the early 1990s in men of all races/ethnicities, although they remain
more than twice as high in blacks as in any other group. Overall, prostate cancer death
rates decreased by 3.5% per year from 2003 to 2012 (American Cancer Society. Cancer
Facts & Figures 2016. Atlanta: American Cancer Society; 2016). These declines are due
to improvements in early detection and treatment. Early prostate cancer usually has no
24
symptoms. With more advanced disease, men may experience weak or interrupted urine
flow; difficulty starting or stopping the urine flow; the need to urinate frequently,
especially at night; blood in the urine; or pain or burning with urination. Advanced
prostate cancer commonly spreads to the bones, which can cause pain in the hips, spine,
ribs, or other areas. There are currently no practices that will guarantee prevention of
prostate cancer; however, there are habits and life style practices that are encouraged as
ways to reduce a man’s risk of acquiring the disease (Figure 3C). As well as some
practices that could potentially increase a man’s risks of getting the disease (Figure 3D).
Early stage prostate cancer (Figure 5) which is localized in the prostate gland is
treatable by surgery and radiation therapy and the prognosis in these patients is very
good.121 Many of these treatments provide men with very little benefit in terms of life
expectancy, but subject them to considerable harm. For instance, one in two is impotent,
one in ten needs to wear pads because of urine leakage and one in ten has back passage
problems. Currently, treating prostate cancer depends on: the stage of the cancer, the
Gleason score, the level of prostate specific antigen (PSA) in the blood stream, the man’s
age and general health, and the side effects of the treatments. According to the Prostate
Cancer Foundation, treatments may include: image guided radiotherapy and intensity
modulated radiotherapy, active surveillance, surgery, external beam radiotherapy,
brachytherapy, hormone therapy, and high intensity focused ultrasound. Most of these
treatments are specific for the localized cancer; once the cancer has escaped from the
prostate, treatment becomes quite difficult and the patients’ quality of life is severely
affected.
25
Figure 5: Stage I, Earliest stage, where the cancer is so small that it cannot be felt on
rectal examination, but is discovered in a prostate biopsy or in prostate tissue that has
been surgically removed to ‘unblock’ the flow of urine (as in a transurethral resection of
the prostate – TURP). Stage II, The tumor can now be felt on rectal examination, but is
still confined to the prostate gland and has not spread. Stage III, The tumor has spread
outside the gland and may have invaded the seminal vesicles. Stage IV, The tumor has
spread to involve surrounding tissues such as the rectum, bladder or muscles of the pelvis
and lymph nodes.
Prostate cancers in later stages of disease metastasize (Figure 6) to other tissues
and bone and pose a significant problem for treatment.121 Prostate cancer cells eventually
break out of the prostate and invade distant parts of the body, particularly the bones and
lymph nodes, producing secondary tumors, a process known as metastasis. Once the
26
cancer escapes from the prostate, treatment is possible, but “cure” as we know it becomes
impossible. Death from prostate cancer results when cancer cells become metastatic after
invading the lymph nodes and blood vessels and migrate to bone.122-123
Figure 6: Transformed metastatic prostate cells escape from the prostate into blood
vessels to eventually invade distant organs.
The most frequent sites of metastasis are lymph nodes and bone; 90% of patients who die
of prostate cancer harbor bone metastases.124-125 Current treatments for metastatic disease
are hormonal therapy and chemotherapy. Hormonal therapies are based on inhibition of
biosynthesis and/or action of androgens.126-127 However, the cancer cells develop resistance
to these treatments resulting in development of castration resistant or hormone refractory
prostate cancers. There is no effective therapy for these cancers which are responsible for
mortality in majority of patients.
27
Metastasis is the cause of most prostate cancer deaths,128 approximately 80% of
metastatic prostate cancers exhibit some degree of bone metastasis.129 The most typical
locations of the metastases are pelvic lymphatic glands, bones and lungs, and very rarely
it metastasizes into a testis.130 Bubendorf et al., in the series of 1589 patients with the
prostate carcinoma, showed that 35% of the patients had hematogenous metastases,
mostly in bones (90%), the lungs (46%) and the liver (25%), while the metastases in the
testis were found only in 0.5% of the cases.130-131
Transforming Growth Factor Beta
In mammals, TGF-β super family consists of over 30 structurally related proteins;
these include 3 forms of TGF-β itself (TGF-β1, TGF-β2, and TGF-β3), 3 forms of
Activins and over 20 Bone Morphogenetic Proteins (BMPs). These growth factors
control a large range of cellular behavior132 including regulating cell growth,
differentiation, and matrix production.133-135 TGF-β is a disulfide-linked homodimeric
protein which is secreted as part of a complex consisting of two units of the large
precursor segment of the TGF-β pro-polypeptide linked in a non-covalent association
with the mature TGF-β dimer.136 This complex is "latent" in the sense that it does not bind
to TGF-β receptors and therefore cannot exert any biological activities associated with
TGF-β.136 The release under physiological conditions of active TGF-β from the latent
complex may be a finely regulated event involving specific proteases.136
The TGF-β isoforms is initially synthesized as a 75-kDa homodimer known as
pro-TGF-β. Pro-TGF-β is then cleaved in the Golgi to form the mature TGF-β
homodimer.137-138 These 25-kDa homodimers interact with latency-associated proteins to
28
form the small latent complex.137-140 In the endoplasmic reticulum, a single latent TGF-β
binding protein forms a disulfide bond with the TGF-β homodimer to form the large
latent complex, allowing for targeted export to the extracellular matrix.138-139 After export,
the large latent complex interacts with fibronectin fibrils and heparin sulfate
proteoglycans on the cell membrane. Eventually, the large latent complex localizes to
fibrillin-rich microfibrils in the extracellular matrix, where it is stored until its
activation;138, 141-142 remaining biologically unavailable until its activation.137-138 Latent TGF-
β is activated by several factors, including proteases,138, 142-143 thrombospondin 1,138, 144
reactive oxygen species,138, 145 and integrins.138, 146-147 These factors release mature TGF-β by
freeing it from the microfibri l- bound large latent complex. This occurs through
liberation from latency-associated proteins, degradation of latent TGF-β binding protein,
or modification of latent complex conformation.138
The three mammalian isoforms of TGF-𝛽 are each encoded by different genes and
share extensive homology (70-80% amino acid sequence identity).94, 148 TGF-β signaling
begins with the binding of activated TGF-β to specialized receptors on the cells
membrane. There are three major classes of TGF-β receptor proteins (TGF-β receptors
types 1-III (abbreviated TβRI, TβRII, and TβRIII, respectively)). TβRI and TβRII are
serine-threonine protein kinases. TβRII contains extra cellular ligand binding domain,
and both TβRI/II contain a single transmembrane domain and (Figure 7) a cytoplasmic
serine threonine kinase domain.94
29
Figure 7: (A) TGF-β type I (TβR-I) and TGF-β type II (TβR-II) are single
transmembrane protein serine/threonine kinases with two kinase inserts. The extracellular
domains are rich in cysteine residues. The carboxyl terminal tail is shorter in the TβR-I
compared to the TβRII. The glycine–serine rich (GS) domain, which regulates the
receptor activation, and the L45 loop (an exposed nine-amino acid sequence between
kinase subdomains IV and V), are only found in TβR-I. A comparison of amino acid
sequences in L45 loop region between activin receptor-like kinase (ALK)1 and ALK5 is
shown below. The two h strands (h4 and β5) that flank the L45 loop are shown as arrows.
(B) Endoglin and betaglycan (TGF-β type III receptors or TβR-III) are single
transmembrane TGF-β accessory receptors that lack an enzymatic motif in their short
intracellular domains. The percentages of identical amino acids in specific regions of the
human endoglin and betaglycan are shown. Their cytoplasmic tails contain many serine
and threonine residues and a putative PDZ domain at the last 3 Carboxy terminal
residues. Proteolytic cleavage site and potential glycosaminoglycan (GAG) side chains
that are rich in heparin sulfate and chondroitin sulfate are indicated. (This figure has been
modified with permission from Miyazono et al.
The kinase domains of the types I and II receptors share only 40% amino acid identity.149
TGF-β signal transduction requires the formation of a TβRI-TβRII heteromeric
30
complex.150-158 Once the ligand is activated, TGF-β signaling is mediated through SMAD
and non-SMAD pathways to regulate transcription, translation, microRNA biogenesis,
protein synthesis, and post-translational modifications.35, 138, 159-160 Although the downstream
effects of TGF-β are heavily context dependent, its signaling is at least partially
conserved in many cell types.33, 138 In the canonical pathway, the TGF-β ligand binds to the
TβRII that recruits the TβRI. These receptors dimerize and autophosphorylate
serine/threonine residues, allowing for the phosphorylation of SMAD2 and SMAD3 by
TβRI. The now activated SMAD proteins dissociate from the SMAD anchor for receptor
activation (SARA) protein, hetero-oligomerize with SMAD4, and translocate to the
nucleus, interacting with myriad transcriptional co-regulators and other factors to mediate
target gene expression or repression.35, 138, 161 TβRIII (or betaglycan), a transmembrane
proteoglycan that binds the TGF-β ligand, whose function is relatively unknown.
Although TβRIII appears to lack a cytoplasmic signaling domain, it appears to have
important roles in development, as well as in regulating TβRI and TβRII.138, 162-164
TGF-β also signals through a number of non-SMAD pathways, including p38
MAPK, p42/p44 MAPK, c-Src, m-TOR, RhoA, RAS, PI3K/Akt, protein phosphatase 2A
(PP2A)/p70s6K, and JNK MAPK.138, 160, 165-169 Additionally, two studies have linked
translational regulation to the cytostatic program governed by TGF-β. The first
mechanism involves transcriptional activation of the translation-inhibiting protein
eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) mediated by the
SMAD signaling pathway,138, 170 whereas the second relies on catalytic inactivation of the
translation initiation factor eEF1A1 (eukaryotic elongation factor 1A1) by TβRI.138, 159
31
Both SMAD-dependent signaling and SMAD-independent signaling play multiple roles
in homeostasis, particularly in the growth and plasticity of epithelial cells. SMAD-
dependent TGF-β signaling induces growth arrest through a number of mechanisms,
including control over various cyclin-dependent kinase inhibitors.138, 171-174 SMAD-
independent mechanisms of TGF-β–induced apoptosis involve DAXX/HIPK2 and
transforming growth factor β associated kinase (TAK1)/TRAF6–dependent p38/JNK
activation.138, 175-176
Transforming Growth Factor Beta (TGF-β) in Prostate Development and Function
TGF-β signaling pathway is a key player in metazoan biology, and its
misregulation can result in tumor development.171 TGF-β made its debut with the rise of
the vertebrates. TGF-β evolved to regulate the expanding systems of epithelial and neural
tissues, the immune system, and wound repair.171 Tied to these crucial regulatory roles of
TGF-β are the serious consequences that result when this signaling pathway
malfunctions, namely tumorigenesis.171 TGF-β superfamily was discovered in a hunt for
autocrine factors secreted from cancer cells that promote transformation.22 However, it
soon became clear that TGF-β and the related BMPs regulate diverse developmental and
homeostatic processes and are mutated in numerous human diseases. Furthermore, TGF-
β-superfamily members such as activins, Nodal, and growth differentiation factors
(GDFs) were shown to control cell fate as a function of concentration, thus defining them
as a key class of secreted morphogens.22 The actions of TGF-β are dependent on several
factors including cell type, growth conditions, and the presence of other polypeptide
growth factors.92 The TGF-β pathway has a complicated role in mediating the ability of
32
cells to participate negatively or positively in growth inhibition, proliferation, replication,
invasion, metastasis, apoptosis, immune surveillance, and angiogenesis.
TGF-β-superfamily members are highly conserved across animals and comprise
the largest family of secreted morphogens.22TGF-β elicits context-dependent and cell
specific effects that often appear conflicting, such as stimulation or inhibition of growth
(Figure 8), apoptosis or differentiation.92 It is puzzling how such a diverse array of
responses can result from binding of TGF-β ligand to a receptor complex that activates a
seemingly straightforward signal transduction scheme dependent on shuttling SMAD
transducer proteins from the receptor to the nucleus.92 This paradox is reflected in the
clinic, where in early stage cancers, levels of TGF-β are positively associated with a
favorable prognosis.138 Yet in advanced tumors, levels of TGF-β in the tumor
33
Figure 8: TGF-β in cancer progression: a.) Normally limits epithelial proliferation. b.)
Loss of TGF-β inhibition leads to hyperplasia and supports transformation. c.) TGF-β
growth response can enhances mesenchymal transition leading to invasion. d.) TGF-β
suppresses T-cell response contributing to escape of immune recognition. e.) TGF-β
displays angiogenic effects d.) TGF-β enhances extravasation and attachment of tumor
cells to tissues of distant sites. e.) TGF-β promotes osteoclast response and bone
remodeling in distant metastasis.
microenvironment are positively associated with tumor size, invasiveness, and
dedifferentiation, making TGF-β a useful prognostic biomarker and predictor of
recurrence after initial or failed therapy.31, 138, 177-179
TGF-β expression has been studied in nearly all epithelial cancers including,
prostate, breast, lung, colorectal, pancreatic, and skin cancers.31, 138 TGF-β acts on normal
prostate epithelial cells and some prostate-cancer cells to inhibit proliferation and induce
34
apoptosis.180-182 TGF-β has also been shown to stimulate E-cadherin expression in
prostate-cancer cells treated with an mTOR inhibitor.183 Prolonged incubation of cells
with TGF-β results in the expression of actin-associated proteins (AAP’s),
tropomyosin,184 and transgelin185 which promote the formation of stress fibers.180 Loss of
TGF-β is also known to promote the formation of a less organized cytoskeleton. Studies
have also shown that TGF-β can suppress or induce PTEN expression, depending on the
Ras/ERK status. A Ras/ERK activated pathway facilitates TGF-β suppression of PTEN
via a SMAD-4 independent signaling pathway186 however, when Ras/ERK is blocked,
TGF-β induces PTEN expression through its classical SMAD-dependent pathway and
stimulates a tumor suppressive response.183, 187 Such a switch is echoed in pancreatic
carcinoma cells, where both activated Ras and PI3K cause TGF-β to down regulate E-
cadherin expression through a SMAD-independent pathway.188 In colorectal cancer higher
TGF-β1 protein expression is associated with increasing T-stage and metastatic disease,
indicating that TGF-β1 is of importance in tumor progression.177 Plasma TGF-β levels are
markedly elevated in men with prostate cancer metastatic to regional lymph nodes and
bone.179 In men without clinical or pathologic evidence of metastases, the preoperative
plasma TGF-β level is a strong predictor of biochemical progression after surgery,
presumably because of an association with occult metastatic disease present at the time of
radical prostatectomy.179
Because perturbation of the TGF-β signaling network has a variety of tumorigenic
effects, its mechanisms must be studied further to identify novel points of convergence
with other pathways and maximize both the clinical efficacy and tumor specificity of
35
future therapies. Through investigation of the TGF-β pathway and its relationship with
other oncogenic factors in the tumor microenvironment, additional strategic points of
convergence can be identified and exploited as a means to prevent or reverse tumor
progression.138 As was already discussed, prostate cancer remains an important clinical
problem, with current therapies being far from adequate, so it is essential to develop new
therapeutic approaches. Understanding of the integration of TGF-β pathway known to be
involved in prostate cancer pathophysiology may be central to the development of
improved pharmacological treatments. A dual role of TGF-β in cancer has long been
noted, but its mechanistic basis, operating logic, and clinical relevance have remained
elusive. What causes TGF-β signaling to be altered in cancer? What steps in tumor
progression may benefit from a faulty TGF-β pathway? When does TGF-β act as a
metastatic signal? And, most importantly, how can any of this knowledge be used to treat
prostate cancer?
Activator Protein-1 (AP-1)
The primary control of eukaryotic gene expression occurs at the level of
transcription where genes may be regulated in response to a specific signal or in a
particular tissue-type.66, 189 Transcriptional control of a gene involves the binding of
regulatory proteins or transcription factors to short, cis-acting DNA sequence elements
located within and near the promoter of a gene.66, 190-192 Following the binding of
transcription factors, the activity of RNA polymerase is modulated in either a positive or
negative manner at the start site of transcription.66 There are several types of DNA
sequence sites to which transcription factors will bind, these include: promoter elements
situated close to or at the start site of transcription which are essential for activation or
36
significant levels of transcription;66, 193-195 regulatory elements situated close to the general
promoter region functioning in an orientation-independent manner;66, 195-198 and
enhancer/repressor elements located at a distance from the transcription start site which
increase or decrease the rate of transcription.66, 199-200
AP-1 Family members
AP-1 is a collective term referring to dimeric transcription factors composed of
JUN, FOS or ATF (activating transcription factor) subunits that bind to a common DNA
site, the AP-1-binding site. The consensus binding site for AP-1 was identified as the
palindromic sequence 5’ TGA/TCA 3’, which was found to be responsive to the phorbol
ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) referred to as the TPA-responsive
element (TRE).66, 201 As the complexity of our knowledge of AP-1 factors has increased,
our understanding of their physiological function has decreased.114 AP-1 is an important
and well-studied transcription factor; the protein components of this transcription factor
are encoded by a set of genes known as “immediate-early” (IE) genes.66, 202 IE genes
couple cytoplasmic biochemical changes, arising from the binding of stimulatory agents
to cell-surface receptors to mediate specific cell responses.66 AP-1 was initially identified
as a DNA-binding activity in HeLa cell extracts that bound to cis-elements within the
promoter and enhancer sequences of the human metallothionein IIA gene and simian
virus 40.66, 203
The transcription factor AP-1 was subsequently found to be comprised of protein
dimers, containing the gene products of members of the JUN (JunD, cJUN, JUNB) and
FOS (FosB, cFos, Fra1, Fra2) gene families.66, 204-206 The JUN protein members form
37
homo- and heterodimeric complexes within their own gene family66, 207-208 and with the
protein members of the FOS gene family.66, 89, 209-211 Unlike the JUN proteins, FOS is unable
to form homodimers and must heterodimerize with JUN proteins in order to
transcriptionally activate AP-1-containing promoter constructs in cells.66, 212-213
Structure
The AP-1 family of transcription factors is a basic leucine zipper (bZIP) dimeric
protein complex of structurally and functionally related members of JUN, FOS, ATF and
musculoaponeurotic sarcoma (MAF) protein families. The basic region or DNA binding
domain (DBD) of bZIP proteins contains positively charged amino acid residues required
for DNA binding activity85 (Figure 9). The leucine-zipper domain (LZD), located
immediately downstream of DBD, contains a heptad repeat of leucine residues. LZD
mediates the dimerization of proteins, bringing two DBDs into juxtaposition, thereby
facilitating the interaction of protein dimers with DNA. Although LZD and DBD are
highly conserved among all AP-1 proteins, their amino (NH2) - and carboxy (COOH)-
terminal regions are quite divergent.
38
Figure 9: Schematic diagram showing the modular structures, dimerization and DNA
binding properties of JUN and FOS proteins. A) the location of various modules is
indicated. TAD, transcription-activating domain; LZD, leucine-zipper domain; DBD,
DNA binding domain; N, amino terminus; C, carboxyl terminus. B) LZD mediates the
dimerization of proteins bringing two DBDs into juxtaposition, thereby facilitating the
interaction of protein dimers with DNA. ATF, activation transcription factor; TRE, 12-O-
tetradecanoylphorbol-13-acetate (TPA)-responsive element; CRE, cyclic AMP-
responsive element. Note that CRE has an extra base (underlined) compared with TRE.
The JUN proteins contain the transactivation domain (TAD) at their NH2-terminal
region, whereas FOS members, except Fra1 and Fra2, possess TADs at their NH2- and
COOH-terminal regions (Figure 9). In many tumors these non-transforming FOS
proteins, especially Fra1 and Fra2, might be involved in the progression of many tumor
types.87, 214 The JUN gene family is similar to each other in their gene and protein
39
structure, particularly in the DNA-binding domain and the leucine zipper regions where
there is 75% amino acid homology with the JUN Family.66, 215 The cFos protein is
evolutionally well-conserved, as its protein sequence retains an overall homology of 97,
94 and 79% between rat and mouse, rat and human, and mouse and chicken cFos
proteins, respectively.66, 216 In addition to cFos other related genes have been identified.
FosB and its naturally truncated form ΔFosB (missing the C-terminal 101 amino acids of
FosB),217 Fos-related antigen-1 (Fra-l), and Fos-related antigen-2 (Fra-2), together with
cFos make up the FOS gene family.66, 211, 217-220 These genes are structurally-related to cFos
in terms of having the same number of exons and introns; however, the size of the
untranslated regions of the genes is variable. The amino acid sequence between each
protein is also conserved, particularly in the basic DNA and leucine zipper regions.66, 219
Similarly to cFos, the protein products of other members of the FOS gene family are
unable to bind to DNA individually, and require dimerization with a JUN protein to form
a functional AP-1 complex.66, 89, 210-211, 217 However, despite their homology, these proteins
display different transcriptional activity.221
Functions
AP-1 is an inducible transcription factor,66 that may regulate different target genes
and thus execute distinct biological functions.114 Various agents have been shown to
induce the expression of the FOS gene family; agents such as serum, growth factors,
neurotransmitters, calcium, phorbol esters, metal ions, UV light and cAMP.66 The
decision if a given AP-1 factor is positively or negatively regulating a specific target gene
is made upon abundance of dimerization partners, dimer-composition, post-translational
40
regulation, and interaction with accessory proteins.222 In vitro studies have shown that
JUN/FOS heterodimers are more stable and have a stronger DNA binding activity than
JUN homodimers.85, 223 The reason for this binding specificity amongst bZIP families can
be attributed to the amino acid combination between the leucine residue repeats within
the leucine zipper region.66, 224 cFos has a highly acidic leucine zipper with a large net
negative charge at neutral pH thus a homodimer formation would not be favored owing to
general electrostatic repulsions between the monomer FOS proteins.66, 224 In addition, the
acidic residues important for specificity are aligned along one face of the helix, causing
intra-helical destabilization. The cJun leucine zipper has a more diffuse, net positive
charge, allowing a JUN homodimer to form. However, the interaction of Jun proteins is
not as stable as a FOS/JUN dimer. As the FOS and JUN monomers are of opposite
charge, they may form a more stable heterodimer together as the interhelical component
of the electrostatic destabilization is relieved.66, 224 Different FOS/JUN complexes also
have different affinities for AP-1 sites, and this is partly attributed to different DNA
sequences around the core AP-1 site.66, 223
AP-1-regulated genes include important regulators of invasion, and metastasis,
proliferation, differentiation and survival, genes associated with hypoxia and
angiogenesis.87, 113, 115 Many oncogenic signaling pathways converge at the AP-1
transcription factor complex. The specific influence of a specific AP-1 protein on a
promoter depends on the dimer partners, the promoter architecture as well as other
transcription factors and co-activators acting on the promoter.113
41
Activator Protein-1 in Cancers and Prostate Cancer
As stated earlier, DNA binding is a necessary prerequisite of transactivation; the
expression of different proteins of the JUN and FOS family is crucial for the activation of
downstream genes regulated by AP-1. AP-1 is known to control the expression of several
target genes that regulate cell cycle (cyclin D, p16), differentiation (myogenin,
involucrin), cell survival (Bcl-2, Bcl-xL, FasL), growth factors (VEGF), and cell
adhesion (VCAM, EDAM-1) and angiogenesis/invasion (MMP’s, uPA, osteopontin,
CD44).82 In the prostate, various members of the AP-1 family have been implicated in the
actions of androgen receptors.225 Activation of cJun has been shown to play a role in the
development of androgen independent prostate cancer; the overexpression of cJun has
been shown to inhibit the expression and function of androgen receptor in human prostate
cancer cells.226 The expression of interleukin-6, which increases in hormone refractory
prostate cancer cell, is dependent on constitutive activation of Fra1 and JunD.227 Jun N-
terminal Kinases (JNKs), which phosphorylate and activate AP-1 proteins have been
shown to be involved in proliferation and tumor growth and survival of prostate cancer
cells.225, 228-229 These studies have underlined the importance of AP-1 proteins in the
development and function of prostate cells and a change in the relative abundance and/or
activities of specific proteins may be involved in development and maintenance of
prostate cancers. However, in spite of these studies, the role of individual AP-1 family of
transcription factors in prostate cancers growth, migration and invasion is far from being
elucidated.
42
Activator Protein-1 in TGF-β Signaling
AP-1 transcription factors contribute to various TGF-β biological responses.230
The promoters of TGF-β1-reponsive genes like plasminogen activator inhibitor-1 (PAI-1)
and cJun contain AP-1 binding sites. Mutation of these AP-1 binding sites which impairs
binding of the AP-1 complex inhibits transcriptional activation of these promoters by
TGF-β1.231-232 SMAD proteins possess DNA-binding activity, but the SMAD4-RSMAD
complexes must associate with additional DNA-binding cofactors in order to achieve
binding with high affinity and selectivity to specific target genes. These Smad partners
are drawn from various families of transcription factors, such as the forkhead, homeobox,
zinc-finger, bHLH, and AP1 families.171, 233-234 These findings suggest that SMAD proteins
and AP-1 complex synergize to activate the TGF-β1-responsive promoters.235 Naso et al
2003 showed that JunB is up-regulated by TGF-β-SMAD signaling and may contribute to
the TGF-β-induced EMT and fibrotic responses.236 Recent studies indicate that SMAD-3
directly binds cJun and cFos of the AP-1 complex and that both SMAD-3 and SMAD-4
bind all three Jun proteins.53 Studies also show that TGF-β1 activates SMAD proteins and
AP-1 complex (JunD: FosB) and that over-expression of their dominant negative forms
inhibits TGF-β1 dependent apoptosis.235 Over expression of FosB enhances SMAD-
dependent transcription of TGF-β1 responsive reporter. JunD: FosB recruits SMAD-
3:SMAD-4 to from AP-1:SMAD complex that binds to the AP-1 binding site, 12-O-
tetradecanoyl-13-acetate- responsive gene promoter element.235 These studies show that
both SMAD proteins and AP-1 complexes play a critical role in TGF-β1-dependent
apoptosis. Our data has shown that TGF-β1 transiently induces mRNA and protein
expression levels of AP-1 components in DU145 and PC3 prostate cancer cell lines, but
43
the role of these inducted AP-1 components in TGF-β1 dependent growth, proliferation,
migration, and invasion remains unknown.
AP-1 Proteins in TGF-β signaling in Cancers and Prostate Cancer
Jun family members (c-Jun, JunB, and JunD) were expressed at different levels
and responded differentially to TGF-β treatment. TGF-β effects on JunD protein levels,
but not mRNA levels, correlated with its effects on cell proliferation. TGF-β induced
significant reduction in JunD protein in RWPE-1 and DU145 cells but not in PC3 cells.
Selective knockdown of JunD expression using siRNA in DU145 and PC3 cells resulted
in significant reduction in cell proliferation, and forced overexpression of JunD increased
the proliferation rate.50 Previously published work in our lab shows that overexpression
of c-Jun and JunB decreased the proliferation rate in DU145 cells. Further studies showed
that down-regulation of JunD in response to TGF-β treatment is mediated via the
proteasomal degradation pathway.50 Thus concluding, specific Jun family members exert
differential effects on proliferation in prostate cancer cells in response to TGF-β, and
inhibition of cell proliferation by TGF-β requires degradation of JunD protein.50
44
CHAPTER 3
MATERIALS AND METHODS
Chemical and Reagents
Recombinant human TGF-β1 (Catalog # HEK293 100-21) was purchased from
Peprotech (Rocky Hill, NJ). The antibodies against FosB (Catalog# 2251S), cFos
(Catalog # SC-52), Fra1 (Catalog # SC-605), and Fra2 (Catalog # SC-604) were
purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX). The anti-β-Actin (Catalog
# A5441) antibody was purchased from Sigma-Aldrich (St. Louis, MO). The anti-mouse
and anti-rabbit immunoglobulins coupled with horseradish peroxidase (IgG-HRP) were
obtained from Promega (Madison, WI). Cell lysis buffer was purchased from Cell
Signaling (Danvers, MA). TRIzol was purchased from Invitrogen (Carlsbad, CA).
Human Prostate Cell Lines and Treatments
All cell lines were obtained from American Type Culture Collection. These
include immortalized prostate luminal epithelial cell line (RWPE1) and prostate cancer
cell lines (LNCaP, DU145, and PC3).
Expression of FOS family members: Prostate cells (RWPE1, LNCaP, DU145 and PC3)
were cultured using established procedures.151, 157, 237 To determine the basal expression of
Fos mRNA and protein levels, RWPE1, LNCaP, DU145 and PC3 cells were seeded at
45
a density 1.0 X 106 cells/dish in a 10 cm petri dish in the appropriate growth media and
cultured at 37°C for 48 h. After 48 h, the cells were washed with ice-cold phosphate
buffered saline (PBS) and lysed in cell lysis buffer containing 20 mM Tris-HCl (pH 7.5),
150 mm NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium
pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 µg/mL leupeptin, and 1 X
protease inhibitor cocktail (Calbiochem, San Diego, California). Protein concentrations
were determined by the Lowry HS assay using the Bio-Rad DC Protein Assay kit (Bio-
Rad Laboratories, Inc., Hercules, CA.) according to the instructions provided by the
manufacturer. The total RNAs were isolated from parallel experiments and were also
used for RT-PCR as described below. To determine the effects of TGF-β1 on FosB, cFos,
Fra1, and Fra2 expression, prostate cancer cells were seeded in six-well plates at a
density of 3.0 X 105 cells per well. Before each experiment DU145 and PC3 cells were
incubated with media supplemented with 1% serum for 2 h followed by treatment with
different doses of TGF-β1 (0, 1, 5, 10 ng/mL) for specific time periods. RNA and
proteins were isolated and quantified.
RNA isolation, cDNA synthesis, and RT-PCR
Total RNAs were isolated from prostate cells using TRIzol (Invitrogen, Carlsbad,
CA.) and the resulting RNA samples were quantified by optical density reading at 260
nm as described previously.238 Total RNAs (2 µg) were reverse transcribed in a 50 µl
reaction mixture containing 0.5 mM dNTP (Fisher Scientific, Pittsburgh, PA), 0.5 mM
dithiotreitol (Bio-Rad, Hercules, CA), 0.5 µg of oligo dT, and 400 U of M-MLV Reverse
Transcriptase (Promega, Madison, WI.) at 37°C for 1.5 h. The reaction was terminated
by heating the samples at 65°C for 5 min and subsequently cooled to 4°C. PCR was
46
performed to detect mRNA levels of FosB1, FosB2, cFos, Fra1, Fra2, and L-19. The PCR
mixture was composed of 0.1 mM deoxynucleotide triphosphates, 0.5 U Taq DNA
polymerase, 10X PCR Buffer with 3 mM MgCl2 and 25 pM of the specific primers in a
total volume of 15 µL. Primer information and the size of specific amplicons for
individual genes are shown in Table 1. L-19 (a ribosomal protein) was used as a loading
control. RNA samples processed without RT and PCR amplified were used as negative
controls. Amplification was performed at 1, initial denaturant 94°C for 2 min; 2, 94°C for
15s; 3, 58°C for 15s; 4, 72°C for 30s, 5, repeating steps 2 and 4 for 35 cycles for FosB1,
FosB2, cFos, Fra1, Fra2, and L-19; 5, final extension 72°C for 2 min. The PCR products
were separated on 1.0-2.0% agarose gels, and viewed under UV.
Western Blot Analyses
Total cellular proteins were prepared from different prostate cell lines and were
analyzed by Western blot as described previously.238 Briefly, cell lysates were mixed with
Laemmeli’s buffer (62.5 mM Tris, pH 6.8, 2% SDS, 5% β–mercaptoethanol, and 10%
glycerol). Individual samples containing 30–35 µg protein were subjected to SDS-PAGE
in 10% gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore
Corp., Bedford, MA). After blocking the membranes with 5% fat free milk in TBST (50
mM Tris, pH 7.5, containing 0.15 M NaCl, and 0.05% Tween–20) (cFos, Fra1, Fra2) or
5% bovine serum albumin (BSA) in TBST (FosB), for 1 h at room temperature, the
membranes were incubated with appropriate dilutions of specific primary antibodies
(1:500 for Fos proteins and 1:5000 for β-actin) overnight at 4°C. After washing, the blots
were incubated with secondary anti-rabbit (for FOS proteins) and anti-mouse (for β-actin)
IgG HRPs (1:20,000) for 1 h. The blots were developed in ECL mixture (Thermo Fisher
47
Scientific Inc., Rockford, IL), and placed inside the Syngene PXi/PXi Touch darkroom
imaging (high resolution, multi-application image analysis systems) (Frederick, MD)
according to the manufacturer’s directions and the density of specific protein bands were
determined using ImageJ image processing and analysis software and values were
normalized using β-actin.
Immunofluorescence of FosB
PC3 cells were seeded at a density of 8.0 x 104 cells/well into six-well plates
containing sterile glass coverslips. The cells were incubated at 37°C and allowed to attach
for 48 h. The media were replaced with fresh media containing 1% FBS for 2 h before
treatment with TGF-β1 (10 ng/ml) for 4 h. At the end of the treatment, media were
aspirated and cells were fixed in 3.7% paraformaldehyde for 20 minutes at room
temperature. The fixative was aspirated and cells were rinsed three times with 1.0 ml 1X
PBS and permeabilized using 0.1% Triton at room temperature. The cover slips were
transferred to an aluminum wrapped 20 cm Petri dish and outlined using a hydrophobic
marker. The cells on the cover slips were blocked in blocking buffer containing 10%
normal goat serum in 1X PBS for 1 h. Blocking solution was aspirated and primary
antibody (1:1000) for FosB was added overnight at 4°C in 1X PBS with 2% normal goat
serum. After washing 5 times in 1X PBS for 10 minutes each, secondary antibody
containing green fluorochrome (Alexa Fluor 488 goat anti-rabbit IgG, Life Technologies,
Carlsbad, CA) was added at room temperature for 1 h in 1X PBS (light sensitive). After
washing, DAPI (3 µg/ml) was added to cells for 20 minutes at room temperature to stain
the nuclei. The cells were rinsed and the cover slips were then mounted on slides. Slides
were kept at room temperature in the dark for 2 h before viewing under inverted
48
florescence microscope. Images were captured using 40 X magnification with an
Axiovision camera of a Carl Zeiss zoom inverted florescence microscope (Carl Zeiss,
Thornwood, NY).
Transfections
Transfection with FosB siRNA
To knockdown endogenous FosB expression, DU145 and PC3 cells were seeded
in six-well plates at the density 1.5 X 105 cells per well in 1.0 mL antibiotic-free normal
growth medium supplemented with 5% FBS. The cells were cultured at 37°C to 60-80%
confluence. Control (scrambled) and FosB specific siRNAs were transfected into DU145
and PC3 cells according to the manufacturers’ instructions. Briefly, transfection complex
were mixed together in a 1:1 ratio (2.5 µL for FosB) of siRNA to transfection reagent in
200 µL of antibiotic-free normal growth medium. The mixture was allowed to incubate at
room temperature in the dark for 20 minutes. During this time the cells were washed once
with 1 mL of siRNA transfection medium, after which the antibiotic-free medium was
mixed with 1% FBS and added to the transfection reagent mixture. The transfection
reagent siRNA duplex was overlaid onto the washed cells, and the cells were incubated
overnight. The medium containing the transfection complex was replaced with complete
medium containing 5% FBS and incubated for 48 h. Cells were trypsinized (0.25%
Trypsin/ 2.21 mM EDTA) for 1 minute and trypsin was neutralized with 3.0 mL of
complete medium. Cells were centrifuged at 1000 RPM 4°C for 5 minutes and re-plated
for biological assays. Western blot analyses were used to confirm FosB protein
knockdown.
49
Transfection with cFos, Fra1 and Fra2 siRNAs
To knockdown endogenous cFos, Fra1 and Fra2 expression, DU145 and PC3
cells were seeded in six-well plates at the density 1.5 X 105 cells per well in 1.0 mL
antibiotic-free normal growth medium supplemented with 5% FBS. The cells were
cultured at 37°C to 60-80% confluence. Control (scrambled) and cFos, Fra1 and Fra2
specific siRNAs were transfected into DU145 and PC3 cells according to the
manufacturers’ instructions. Briefly, transfection complex were mixed together in a 1:1
ratio (6 µL for cFos, Fra1 and Fra2) of siRNA to transfection reagent in 200 µL of
antibiotic-free normal growth medium. The mixture was allowed to incubate at room
temperature in the dark for 20 minutes cells were treated as previously described above.
After being transiently transfected to silence cFos, Fra1 and Fra2 DU145 and PC3 cells
were used in cell proliferation and migration assays.
Transfection with cJun siRNAs
siRNA was used to knockdown endogenous cJun expression, PC3 cells were
seeded in six-well plates at the density 1.5 X 105 cells per well in 1.0 mL antibiotic-free
normal growth medium supplemented with 5% FBS. The cells were cultured at 37°C to
60-80% confluence. Control (scrambled) and cJun specific siRNAs were transfected into
PC3 cells according to the manufacturers’ instructions. Briefly, transfection complex
were mixed together in a 1:1 ratio (6 µL for cJun) of siRNA to transfection reagent in 200
µL of antibiotic-free normal growth medium. The mixture was allowed to incubate at
room temperature in the dark for 20 minutes cells were treated as previously described.
After being transiently transfected to silence cJun, PC3 cells were used in cell migration
assays.
50
Cell Proliferation Assays
MTT Assays
After transient transfection, the cells were counted and seeded into 96-well plates
at a density of 5 X 103cells/well and treated with 10 ng/mL of TGF-β1 in the presence of
1% FBS for 72 hours. Cell viability was measured using 3-(4, 5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide (MTT) assay. MTT assays were performed using Cell
Titer 96 Non-radioactive Cell proliferation assay (Promega, Madison, WI) following the
manufacturer’s instructions.
MTS Assays
After transient transfection, the cells were counted and seeded into 96-well plates
at a density of 5 X 103cells/well and treated with 10 ng/mL of TGF-β1 in the presence of
1% FBS for 72 hours. Cell viability was measured using 3-(4, 5-dimethylthiazol-2-yl)-5-
(3-carboxymethoxyphenyl) -2-(4-sulfophenyl)-2H-tetrazolium inner salts (MTS) assay.
MTS assays were performed using Cell Titer 96 AQueous One Solution Cell proliferation
assay (Promega, Madison, WI) following the manufacturer’s instructions.
Total Cell Number Assays
After DU145 and PC3 prostate cancer cells were transiently transfected to silence
the Fos family proteins (cFos, Fra1 and Fra2) cells were trypsinized using 400 µl of
trypsin and neutralized using 3000 µl of MEM and centrifuged at 5000 rpm, 4ºC for 5
minutes to remove trypsin. The cells were then re-suspended in 1000 µl MEM in a 1.5 µl
Eppendorf tubes as previously described and counted using the hemocytometer. Media
was gently pipetted to ensure the cells are evenly distributed. Before the cells have a
chance to settle, 20 µl of cell suspension was removed using a sterile pipette and placed
51
into both chambers on the hemocytometer glass slide underneath the coverslip, allowing
the cell suspension to be drawn out by capillary action. An Oxioskop 2 Plus Ziess light
microscope was used to focus on the grid lines of the hemocytometer with a 10X
objective. Cells were then counted using a hand tally counter. A counting system was
employed whereby cells were only counted when they are set within a square or on the
right-hand or bottom boundary line. The hemocytometer was moved to the next set of 16
corner squares and carry on counting until all 4 sets of 16 corners are counted. This was
done twice for each treatment allowing a total of 8 sets of 16 squares per treatment. The
average of each set of 16 corner squares were taken and multiplied by 10,000 (104). Each
treatment was done at least three times using different cell preparation each time and
average displayed in a representative figure that showed the number of cells represented
on the Y axis and varying treatments on the X axis.
Cell Migration Assays
After the transfections, in vitro cell migration assays were performed using 24-
well transwell inserts (8 µm) as previously described.239-240 Chemoattractant solutions were
made by diluting TGF-β1 (10 ng/ml) or EGF (10 ng/ml) into MEM for DU145 and PC3
cells supplemented with 0.2% BSA. The results were expressed as migration index
defined as: the average number of cells per field for test substance/the average number of
cells per field for the medium control. The experiments were conducted at least three
times using independent cell preparations.
Cell Invasion Assays
After transfection, the invasive behavior of PC3 cells was measured using the BD
BioCoat Matrigel Invasion inserts.49 Cell culture inserts (VWR International, Bridgeport,
52
NJ) were coated with 50 µl of 1:4 Matrigel/Medium dilutions (BD Sciences, San Jose,
CA) and allowed to solidify at 37°C for 1 h. Cells were resuspended (5.0 X 104 cells/ml)
in MEM and 0.1% FBS and 500 µl of cell suspension was added to each insert.
Chemoattractant solutions were made as described above with TGF-β1 and EGF into
MEM supplemented with 0.1% FBS. Matrigel and non-invading cells were removed by
scrubbing. Invading cells in the membrane were fixed in 3.7% paraformaldehyde and
stained with DAPI. Pictures were taken from five different fields for average number of
invading cells to be determined. The results were expressed as an invasive index defined
as: the average number of cells per field for test substance/the average number of cells
per field for the medium control. The experiments were conducted at least three times
using independent cell preparations.
Co-immunoprecipitation
3.0 X 106 PC3 cells were plated in 10 cm petri dish with total volume of 10 ml
MEM supplemented with 5% FBS and allowed to incubate overnight at 37ºC. The MEM
was then aspirated and replaced with fresh MEM supplemented with 1% FBS for 2 hours
before treatment. The cells were then treated with 10 ng/ml of TGF-β1 for 4 hours and
then harvested under non-denaturing conditions. Briefly, MEM was removed and cells
were rinsed once with ice cold 1X PBS. The PBS was removed and replaced with ice
cold cell lysis buffer (containing 20 mM Tris-HCl (pH 7.5), 150 mm NaCl, 1 mM
Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-
glycerophosphate, 1 mM Na3VO4, 1 µg/mL leupeptin, and 1 X protease inhibitor cocktail
(Calbiochem, San Diego, California)) and placed on ice for 10 minutes. The cells were
then scraped off the plates and transferred to a 2.0 ml micro-centrifuge tube. The lysates
53
were then sonicated three times 5 seconds each whilst still on ice. Cells suspension was
then centrifuged for 10 minutes at 14,000 X g, 4ºC and the supernatant was transferred to
a new tube. The cell lysates were pre cleaned to reduce nonspecific binding of Protein by
combining 400 µl of cell lysates with 40 µl of protein A sepharose beads and incubation
at 4ºC for 45 minutes without rotation shaker. The cells lysates were then spun for 10
minutes at 4ºC and the supernatant transferred to a fresh tube. FosB primary antibody was
added (1:50 dilution) to 400 µl of the cell lysates and incubated with gentle rocking
overnight at 4ºC. 40 µl of protein A sepharose beads were added to cell lysates and
incubated overnight at 4ºC with gentle shaking. Tubes containing cell lysate and protein
A were then centrifuged at 4ºC for 30 seconds. The supernatants were transferred to fresh
tubes. The pellets were washed 5 times with 500 µl of 1X cell lysis buffer and kept on ice
during washes. The pellets were re-suspended in 40 µl of 2X SDS sample loading buffer,
vortexed and the micro centrifuged for 30 seconds at 4ºC. The samples were then heated
95-100ºC for 5 minutes and centrifuged for 1 minute at 14,000 X g. After which the
samples were loaded on SDS-PAGE gel and analyzed using Western Blot.
Statistical analysis
All experiments were repeated at least three times using a different cell
preparation. ANOVA, Duncan’s modified multiple range and Student-Newman-Keuls
tests were employed to assess the significance of differences between treatment groups.
54
CHAPTER 4
RESULTS
Expression of FOS family members in prostate cancer cell lines
We initially screened four human prostate cell lines, RWPE1 (normal prostate
epithelial cells), LNCaP, DU145 and PC3 (prostate cancer cell lines) for expression of
FOS family members. Levels of mRNA for FosB, cFos, Fra1 and Fra2 were determined
using RT-PCR, with L-19 used as control (Figure 10A). Fra2 mRNA was robust in all
four cell lines, Fra1 and FosB mRNA were highly expressed in all cell lines except in
LNCaP, which also had very low mRNA levels of cFos ΔFosB was highly expressed in
all cells with only moderate expression in RWPE1 cells (Figure 10A). Western blot
analyses was performed to determine the relative protein abundance of FosB, cFos, Fra1
and Fra2 (Figure 10B). Fra1 protein levels were very low in all prostate cancer cell lines
(LNCaP, DU145, and PC3) with slightly higher levels in RWPE1 cells (Figure 10B).
Fra2 protein levels were relatively high in RWPE1, DU145 and PC3 but were not
detectable in LNCaP cells. cFos showed moderate expression in RWPE1, DU145 and
PC3 cells but was very low in LNCaP cells. FosB and ΔFosB protein levels were high in
RWPE1 and PC3 cells but low to moderate in LNCaP and DU145 cells respectively
(Figure10B) shown below.
55
Figure 10: FOS family basal expression- Steady state mRNA levels of FOS (FosB, cFos,
Fra1, and Fra2) mRNA and protein expression. A, Total RNA’s were isolated and semi
quantitative RT-PCR was performed to determine the mRNA levels of FosB, cFos, Fra1,
and Fra2 in prostate cells. L-19 was used as an internal control. B, western blot analysis
of FosB, cFos, Fra1, and Fra2 in prostate cells. β-actin was used as a loading control.
56
TGF-β1 effects on FOS protein expression and nuclear accumulation in prostate
cancer cells
DU145 and PC3 cells were treated with exogenous TGF-β1 (10 ng/ml) for 0, 1, 2,
and 8 h. TGF-β1 induced an increase in the mRNA levels of FosB (P<0.05) in both cell
lines but had little effect on the mRNA levels of the other FOS family members (Figure
11A). At the protein levels, TGF-β1 induced an increase in the levels of FosB (P<0.05)
protein in both cell lines and an increase in Fra2 (P<0.05) protein levels was observed
only in PC3 cells. TGF-β1 had no effect on the levels of cFos and Fra1 in both cell lines
(Figure 11B). Spot densitometry analysis confirmed TGF-β1 effects on FosB and Fra2
protein levels in DU145 and PC3 cells in a time-dependent manner (Figure 11C). TGF-
β1 induction of FosB was dose dependent (Figure 11D). Immunofluorescence of FosB in
PC3 cells showed that treatment with TGF-β1 induced increased expression and nuclear
localization of FosB (Figure 11E). Inhibition of TGF-β receptors as well as Smad3
abrogated TGF-β1 ability to increase FosB protein expression (Figure 11F); however
inhibitors against PI3K and MAPK had no effect on TGF-β1 ability to increase FosB
protein expression (Figure 11F).
57
Figure 11: A, RT-PCR analysis of FosB, cFos, Fra1, and Fra2 mRNA levels in DU145
and PC3 prostate cancer cells after exposure to exogenous TGF-β (10 ng/mL) for
different times. B, western blot analysis of FosB, the FosB antibody used recognizes both
full length FosB (higher molecular weight band) and ΔFosB (lower molecular weight
band), cFos, Fra1, and Fra2 protein levels DU145 and PC3 prostate cancer cells after
exposure to exogenous TGF-β1 (10 ng/mL) for different times. C, Band density analysis
of FosB and Fra2 in DU145 and PC3 cells after treatment with TGF-β1 for 2 and 8 hours.
Each band density was normalized by density of β-actin bands. Each bar represents the
Mean ± SD from 3 independent experiments. “a and b” denote significant differences
(P<0.05) from untreated controls.
58
Figure 11: D, The Dose dependent effects of TGF-β1 on expression of FosB; Western
blot analysis of FosB in prostate cancer cells DU145 and PC3 after treatment with
varying concentrations of exogenous TGF-β1 (0, 1, 5, 10 ng/mL) for 4 hours. E,
Immunofluorescence, TGF-β1 activation of FosB in PC3. Cells were treated with
exogenous TGF-β1 (10 ng/mL) for 0, and 4 hours. F, DU145 and PC3 cells were treated
with inhibitors for TGF-βRI/II, Smad3, PI3K and MAPK.
FosB knockdown on TGF-β1-mediated cell proliferation, migration and invasion
Next we determined the role of FosB in TGF-β1-induced cell proliferation,
migration and invasion in prostate cancer cells. A transient knock down of FosB was
carried out using siRNA specific to FosB, followed by a MTT proliferation (Figure 12A,
B), trans-well inserts migration assays (Figure 13A, B) and Matrigel invasion assays
(Figure 14). Knock down of FosB had no effect on cell proliferation in DU145 and PC3
cells (Figure 12 A, B) however; there was a significant decrease (P<0.05) in cell
migration (DU145, PC3) and cell invasion (PC3: P<0.05) in response to TGF-β1 and
59
EGF in these cells (Figure 13A, B and Figure 14). Our data also suggests that FosB
knockdown reduced both TGF-β1-and EGF- induced cell invasion but had no significant
effect on the basal invasive potential of these cells (Figure 14).
Figure 12: Effects of FosB knock down on TGF-β1-induced cell proliferation- DU145
and (A) PC3 (B) cells were transfected with siRNA to transiently silence FosB followed
by an in vitro proliferation assay.
60
Figure 13: Effects of FosB knock down on cell migration- Prostate cancer cells DU145
(A) and PC3 (B) were pretreated with siRNA against FosB for 72 hours. Western blots
were used to confirm knock down of endogenous FosB (inserts). DU145 and PC3 cells
were pretreated with siRNA against FosB, followed by treatment with 10 ng/mL of
exogenous TGF-β1 and 10ng/ml EGF migratory behavior were measured using transwell
insert migration assay. Each bar represents Mean ± SEM from three independent
experiments. Different letters designate statistically significant (P<0.05) differences
among different treatments.
61
Figure 14: Effects of FosB knock down on cell invasion -PC3 cells were pretreated with
siRNA against FosB, followed by treatment with 10 ng/mL of exogenous TGF-β1 and 10
ng/ml EGF invasive behavior were measured using and Matrigel in vitro invasion assay.
Insert shows western blot used to confirm FosB knock down. Each bar represents Mean ±
SEM from three independent experiments. Different letters designate statistically
significant (P<0.05) differences among different treatments.
FOS Family Members role in Prostate Cancer Cell Growth and Proliferation
After finding out that FosB increased protein expression in DU145 and PC3
prostate cancer cells in response to TGF-β1 stimulation had no effect on prostate cancer
cell proliferation; the other FOS family members (cFos, Fra1, and Fra2) were transiently
silenced using siRNA specific to each family. This was followed by cell count and MTS
proliferation assay. Our results indicated that transiently silencing cFos and Fra1 had no
effect on cell number in DU145 prostate cancer cells (Figure 15A). Our data also showed
62
that cFos knock-down increased (P<0.05) cell number in PC3 cells and Fra1 knock down
had no effect on cell number in these cells (Figure 15B). Our data showed that TGF-β1
increases the expression of Fra2 (Figure 11B, C) protein expression in PC3 prostate
cancer cells only. In order to determine the role of this increased Fra2 protein expression
Fra2 was transiently silenced in both DU145 and PC3 prostate cancer cells followed by
cell count and MTS proliferations assays. The cell count data (not shown) indicated that
Fra2 knock-down in PC3 cells results in decrease in cell number, MTS data supported the
data seen by cell counting showing that Fra2 knock-down in PC3 cells resulted in
decreased (P<0.05) cell proliferation (Figure 16B). On the other hand knock-down of
Fra2 had no effect on cell count or cell proliferation in DU145 cells in the presence or
absence of TGF-β1 (Figure 16A). Our data also showed that transiently knocking down
FOS family members had no effect on cell morphology in PC3 prostate cancer cells
(Figure 17).
63
Figure 15: A) DU145 cells were transfected with siRNA to transiently silence cFos and
Fra1 followed by an in vitro proliferation assay. Insert western blot image confirming
cFos and Fra1 knock down. B) PC3 cells were transfected with siRNA to transiently
silence cFos and Fra1 followed by an in vitro proliferation assay. Different letters
designate statistically significant (P<0.05) differences among different treatments. Insert
western blot image confirming cFos and Fra1 knock down.
64
Figure 16: A) DU145 cells exposed to siRNA specific for Fra2 followed by stimulation
with exogenous TGF-β1 (10ng/ml) for 72 hours. Inserts show western blot analysis
confirming Fra2 knock down and 96 well plate layout of treated cells. B) PC3 cells
exposed to siRNA specific for Fra2 followed by stimulation with exogenous TGF-β1
(10ng/ml) for 72 hours. Inserts show western blot analysis confirming Fra2 knock down
and 96 well plate layout of treated cells. Different letters designate statistically significant
(P<0.05) differences among different treatments.
65
Figure 17: PC3 cells were transiently transfected to silence FosB, cFos, Fra1, and Fra2,
morphology images were obtained using Ziess microscope at X10 magnification.
TGF-β1 Effect on AP-1 dimerization
In an attempt to determine which JUN protein could function as FosB dimer
partner responsible for its effects on in cell migration; PC3 prostate cancer cells were
seeded at a density of 3 million cells per 10 cm dish and stimulated with TGF-β1 for 4
hours, the cells were lysed with cell lysis buffer as described previously and lysates used
to perform a co-immunoprecipitation assay to determine if FosB dimerization with JUN
proteins was regulated by the presence of TGF-β1. Our data showed that FosB
dimerization with JunD is minimal and not influenced by TGF-β1 (Figure 18). The effect
of TGF-β1 on FosB: JunB dimerization is currently being investigated. The most
66
interesting finding is that TGF-β1 reduced FosB: cJun dimerization (Figure 18). This led
to an interest in determining whether cJun has a role in prostate cancer cell migration.
Figure 18: PC3 cells were stimulated with TGF-β1 followed by co-immunoprecipitation
assay to determine the effect of TGF-β1 stimulation on FosB dimerization with JUN
proteins.
The Role of cJun Protein in Prostate Cancer Cell Migration
Our data showed that FosB is essential for basal, TGF-β1-and EGF-induced cell
migration to occur. Previous studies have shown that FOS proteins must function as
dimers more specifically heterodimers and that dimerization is a prerequisite for nuclear
translocation and thus activation of AP-1 dimer complex 71. This finding as well as our
data indicating that TGF-β1 stimulation decreases cJun: FosB dimerization led to a new
found interest in the role of cJun in FosB effects, specifically in TGF-β1 signaling and
prostate cancer cell migration. cJun was transiently silenced in PC3 cells using siRNA
67
specific for cJun followed by transwell migration assay. Our data showed that cJun
knock-down led to significant increase (P<0.05) in basal, TGF-β1-and EGF-induced cell
migration (Figure 19). Using DU145 prostate cancer cells that were stably transfected in
our lab to over-express cJun; we determined that over-expression of cJun leads to a
significant decrease (P<0.05) in basal cell migration (Figure 20). We are currently
working on transiently over-expressing cJun in PC3 prostate cancer which will be
followed by cell migration assays, as well knocking down JunB and JunD and
determining their effects if any on prostate cancer cell migration.
Figure 19: PC3 cells transiently transfected to knock down cJun and stimulated with 10
ng/ml of TGF-β1 and EGF followed by transwell migration assay, insert showing western
blot image confirming cJun knock down. Different letters designate statistically
significant (P<0.05) differences among different treatments.
68
Figure 20: DU145 cells stably transfected to over express cJun and stimulated with 10
ng/ml of EGF followed by transwell migration assay. Different letters designate
statistically significant (P<0.05) differences among different treatments.
69
CHAPTER 5
DISCUSSION
In this study, we demonstrate for the first time the role of FOS transcription
factors in migration and invasion of prostate epithelial cancer cells and the role of cJun in
prostate cancer cell migration. To determine the role of FOS proteins in prostate cancer
cell proliferation migration and invasion, we performed two types of experiments: first,
the effect of TGF-β1 on the Fos family expression levels were determined by western
blot analysis using different doses and varying times of exposure to TGF-β1; second,
FOS knock-down was used to determine their roles in TGF-β-regulated prostate cancer
cell proliferation, migration, and invasion. The key findings in this study are that 1) TGF-
β1 induces and increased expression of FosB in prostate epithelial cancer cells, 2) FosB is
essential for migration and invasion to occur in prostate cancer cells, and 3) FosB is
required for TGF-β1-and EGF-induced cell migration and invasion, 4) TGF-β1 induces
increased protein expression of Fra2 in PC3 cells only, 5) Fra2 is necessary for basal and
TGF-β1 induced cell proliferation in PC3 cells, 6) TGF-β1 decreases FosB : cJun
dimerization, 7) cJun over expression inhibits prostate cancer cell migration, 8) cJun
70
Knockdown increases cell migration.AP-1 family of transcription factors are a part of the
complex immediate early genomic response of a variety of cells to transmembrane
signaling agents.217 Additional complexity results from the variety of possible Jun dimers
and the JUN-FOS heterodimers and from potential dimer formation between JUN or FOS
and other leucine zipper proteins.217 JUN and FOS proteins share extensive homology
within the leucine zipper and basic domains. However, despite their homology, these
proteins display different transcriptional activity.221 The FOS proteins contribute distinct
functions towards the activity of the AP-1 heterodimers. For example, c-Fos can both
activate and repress transcription,79 the full-length Fos B is a transcriptional activator241
and a naturally occurring short form of FosB inhibits AP-1 transactivation.242 The Fos-
related antigens, Fra-1 and Fra-2 lack functional transactivation domains and are poor
transcriptional activators.242 We believed that an alteration in the composition of AP-1
either directly or indirectly regulates cell growth and motility, which in turn pushes the
normal cell into pre-malignant or malignant state. Therefore we analyzed the effect of
TGF-β1 on Fos mRNA and protein expression in both normal as well as different
prostate cancer cell lines. The most interesting observation was an immediate increase in
FosB expression both at the mRNA and the protein levels in prostate cancer cells as well
as increased Fra2 protein expression in PC3 cells only.
TGF-β super family signaling is well known as a key regulator of many biological
processes31, 49, 230, 243 including differential effects on cell proliferation and migration in
prostate cancer cells.31, 49, 243 These differential effects of TGF-β during different stages of
cancer progression presumably depend on selective loss or acquisition of specific
71
intracellular signals that are required to elicit different biological responses to TGF-β. A
loss of TGF-β receptors and/or Smad proteins has been shown to result in TGF-β
resistance in cancer cells.237, 244-245 However, most cancer cells retain classical TGF-β
signaling components throughout cancer progression but modify or recruit additional
signaling pathways to exert novel or different biological effects.237 Our data shows that
TGF-β1 increases FosB expression in prostate cancer cells, which, in turn, mediates its
effects on migration and invasion but does not play a role in TGF-β1 effects on cell
proliferation. Thus TGF-β1 induction of FosB may represent a shift in intracellular
signaling involved in the escape from inhibition of proliferation to the stimulation of
more migratory and invasive behavior in advanced stages of prostate cancer.246 TGF-β1
reduces the dimer formation between cJun and FosB; our data demonstrates that the
presence of cJun contributes to inhibition of cell migration; this information could shed
light on the dual role of TGF-β1 prostate cancer cell progression, a role that could involve
the regulation of AP-1 dimer formation as least in the case of cell migration.
While essential to normal development and homeostasis, the process of cellular
migration is also a trait essential for metastasis. Enhanced migration is key across the
metastatic cascade and is involved in the initial scattering of cells and migration from the
primary tumor.189 Numerous proteins and pathways have been implicated in altering the
migratory potentials of cancer cells and therefore their aggressive nature. Given its
essential role in cancer progression, treatments that inhibit cell migration or such
proteins/pathways involved in enhancing cellular motility represent an attractive strategy
for controlling metastatic dissemination.189 Because Fra1 and Fra2 exhibit a lack of trans-
72
activating domain as seen in cFos and FosB, they might exert inhibitory functions on
tumor growth. Yet recent data point to a positive effect of Fra1, and partly Fra2, on tumor
progression in many tumor types.191, 214 Our data suggests that Fra2 plays an important role
in cell proliferation of aggressive prostate cancer cells but does not have the same effect
on prostate cancer cells in the early stages of development. In contrast to the bulk of data
on the function of cFos and Fra1, far less is known about the role of FosB and its smaller
splice variant ΔFosB which is often expressed more strongly than Fra1 in clinical cancer
tissues.214, 247 Although, the FOS family of proteins has been extensively studied as
immediate early genes, the role of FosB in cancer cell proliferation and migration and
invasion has not been previously investigated.246 There are also no studies demonstrating
the role of cJun in prostate cancer cell migration. Our data shows that transient silencing
of FosB with or without the presence of TGF-β1 has no effect on prostate cancer cell
proliferation but significantly reduces cell migration and invasion. Numerous studies
have demonstrated that TGF-β1 induces the migration and invasion of prostate cancer
cells; however, we show in this study that TGF-β1 is unable to induce prostate cancer cell
migration and invasion without FosB. The data also suggests that epidermal growth
factor (EGF), a potent mitogenic factor that plays an important role in the growth,
proliferation and differentiation of numerous cell types is unable to induce migration and
invasion in prostate cancer cells in the absence of FosB; further confirming that FosB
does indeed have a major role in migration and invasion of prostate cancer cells. Thus the
differences in migratory and invasive behavior observed in different stages of prostate
cancer progression can be due to AP-1 (specifically FosB) activation. FosB may have a
73
role in the aggressive phenotype observed in prostate cancer, thus inhibition of FosB
activity may serve as a therapeutic tool in the management of prostate cancer.
TGF-β1 is known to switch from being a tumor suppressor in early stage prostate
cancer to becoming a tumor promoter in the later stages of the disease. Our data suggests
that TGF-β1 is unable to induce and increase in cell proliferation without the presence of
Fra2 in PC3 cells which is used in this study to represent an advanced stage prostate
cancer. This further supports the idea of AP-1 proteins playing essential roles in TGF-β1
induced prostate cancer cell progression.
Our data also suggests that TGF-β1 is able to reduce dimer formation between
cJun which we have shown to be necessary for inhibition of prostate cancer cell
migration and FosB which we also shown to be essential for prostate cancer cell
migration to occur. This study has further implicated that targeting AP-1 proteins could
serve as an alternative therapeutic target in treating prostate cancer.
74
CHAPTER 6
CONCLUSION
In conclusion, our results obtained using human prostate cancer cell lines suggest
that the transcription factors FosB, Fra2 and cJun may be important regulators of TGF-β1
effects on proliferation, migration and invasion in human prostate cancer cells. The
functions of AP-1 proteins have been known to be modulated in four major ways: 1)
changes in protein expression, 2) variations in dimer partners, 3) changes in subcellular
localization and 4) changes in phosphorylation. Our data indicates that TGF-β1 is able to
regulate AP-1 by varying their expression, subcellular localization and dimerization. This
was seen as an increase in FosB and Fra2 protein expression, an increase in FosB nuclear
localization, and a decrease in FosB: cJun dimerization as a result of prostate cancer cell
stimulation with TGF-β1. Though still elusive, these studies may help to further
understand TGF-β1 dual role in prostate cancer progression. Further studies are needed to
decipher the Jun protein partner that is influenced by the presence of TGF-β1 to bind to
FosB leading eventually to cell migration and invasion. Also, since TGF-β1 does not
increase cell proliferation in PC3 cells it still remains unclear as to the specific role of
Fra2 protein increase in PC3 cells stimulated with TGF-β1, and how this increased
75
expression contribute to PC3 cells being insensitive to the growth inhibitory effects of
TGF-β1.
Further studies are also needed to completely decipher the role of cJun in
FosB/TGF-β1 induced cell migration. Further study of the roles of FosB, Fra2 and cJun
in prostate cancer carcinogenesis, especially in vivo, will be of great importance and will
probably open new perspectives for therapy.
76
APPENDIX A
Table 1
77
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