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Activator Protein-1 in Transforming GrowthFactor-Beta Effects on Prostate Cancer CellProliferation, Migration, and InvasionCachetne S.X. BarrettClark Atlanta University, cachetne.barrett@students.cau.edu

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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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