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Study of bone morphogenetic protein-2 and stromal cell derived factor-1 in prostate cancer

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Study of bone morphogenetic protein-2 and stromal cell derived factor-1 in prostate cancer

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Content STUDY OF BONE MORPHOGENETIC PROTEIN-2 AND STROMAL CELL
DERIVED FACTOR-1 IN PROSTATE CANCER

by
Azadeh Fata

__________________________________________________________________

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR AND EXPERIMENTAL PATHOLOGY)

May 2012

Copyright 2012 Azadeh Fata

ii
TABLE OF CONTENTS

List of Figures .................................................................................................................... iii

Abstract ............................................................................................................................ iv

Introduction ......................................................................................................................... 1

Hypothesis and Rationale ................................................................................................... 7

Material and Methods ......................................................................................................... 9

Table 1: Primer sequences for qRT-PCR ......................................................................... 12

Results ........................................................................................................................... 14

Discussion ......................................................................................................................... 23

References ......................................................................................................................... 30

iii
LIST OF FIGURES

Figure 1. Potency of the lot of BMP-2 used in this work ................................................. 14

Figure 2. BMP-2 stimulates SDF-1α secretion in AD-CAF and CRPC-CAF cell cultures
........................................................................................................................... 16

Figure 3. Detection of CXCR4 expression in two different types of prostate epithelia cell
cultures: E8 and cE1 .......................................................................................... 17

Figure 4. cE1 and E8 cell growth in medium containing 0.5% serum was determined by
counting the cell number every 2 days in the absence or presence of 300ng/mL
of SDF-1 α recombinant protein ....................................................................... 18

Figure 5. Light microscopy pictures illustrate the morphology of cE1 and E8 cells after
treatment with 200ng/mL of BMP-2 for 24 hr. ................................................. 19

Figuer 6. Representative light microscopy pictures to indicate increased motility into a
wound line of E8 cells which were exposed to 100ng/mL of SDF-1α relative to
the control cells ................................................................................................. 20

Figure 7. Representative light microscopy pictures to indicate increased motility into a
wound line of cE1 cells which were exposed to 100ng/mL of SDF-1α relative to
the control cells ................................................................................................. 21

Figure 8. Migration rate of E8 and cE1 cells into the wound line in μm/hr. in the
presence of 100ng/ml of SDF-1α and absence of SDF-1 α. .............................. 22

Figure 9. BMP-2 stimulates SDF-1 secretion and induces Smad phosphorylation in CAF-
1 cell. ................................................................................................................. 27

Figure 10. Proposed mechanism for the role of PTEN in CXCR4-mediated signaling ... 28

Figure 11. Schematic representation of CXCL12-induced CXCR4 activation in cancer
cells in the bone microenvironment ................................................................... 29

iv
ABSTRACT
The focus of this study is to better understand the role of bone morphogenetic protein-2
(BMP-2), and stromal cell-derived factor-1 (SDF-1) that occur in the prostate cancer
tumor microenvironment with respect to interactions between malignant epithelial cells
and the associated fibroblastic cells. The research concerns two distinct phases of the
disease: androgen-dependent primary tumor (AD cancer) and the post-castration
androgen refractory tumor development-the castration resistant prostate cancer (CRPC).
The model systems used are E8 (primary tumor), and cE1 (CRPC tumor) malignant
epithelial cell lines, and primary cultures of cancer-associated fibroblasts, derived from
primary tumor (AD-CAFs) or CRPC tumor (CRPC-CAFs). All cellular materials have a
homologous origin from the conditional Pten deletion mouse model of prostate cancer.
First, it is shown that all CAFs, AD or CRPC in origin, are responsive to BMP-2 induced
stimulation of SDF-1 expression. While the level of induction varies from one AD-CAF
to another, a single CRPC-CAF tested exhibits a robust induction of SDF-1 by BMP-2.
Second, the constitutive level of secreted SDF-1 is significantly higher in CRPC-CAF as
compared to AD-CAFs. In contrast to these observations on SDF-1 induction, the levels
of the cognate receptor CXCR4 appears to be low in CRPC cancer cells (cE1) in relation
to that in primary tumor cells (E8). This opposite spectrum of ligand and receptor
expression may, perhaps, be a mode of operation to regulate tumor growth in the prostate
tumor microenvironment. Third, the increased level of CXCR4 in E8 cells is
demonstrated to correlate well with their responsiveness to SDF-1 for proliferation or
migration potentials. Finally, it is possible that the high levels of SDF-1 produced by
CRPC-CAFs may have other significant role, such as, in cancer cell invasion and tumor

v
angiogenesis that are critical for the recurrent tumor growth and metastasis. These clues;
however, remain to be better developed in the future.
Taken together this report sets-up a new oncogenic property of BMP-2- SDF-1 axis in
two different types of the prostate tumors, the primary and the recurrent prostate cancer,
introducing an interesting aspect of heterotypic cell interaction potentially critical in
prostate cancer.

1
INTRODUCTION
“Tumors are wounds that do not heal”. This statement implies that certain types of cells
which play major role in angiogenesis and response to injury, such as endothelial cells
and fibroblasts, are also involved in progression, growth, and spread of cancers. Prostate
adenocarcinoma is usually characterized as a slow progressing tumor. It develops in an
orderly fashion from prostate intraepithelial neoplasm (PIN) lesions into adenocarcinoma.
Since prostate cancer is a gradual process, many PIN lesions may form and never
progress to become an advanced tumor. However, if the tumor starts to spread the
treatment of choice will involve hormone therapy. Hormone therapy is the depletion of
androgens in the tumor which results in the regression of androgen dependent tumor
growth. However most of the time the reoccurrence of the tumor will occur resulting in
the growth of hormone refractory prostate cancer (HRPC), which frequently leads to
metastasis at which point the prognosis for the patient is not good.
Using hormones as a therapeutic tool for cancer treatment is an example of how a change
in tumor microenvironment could affect tumor progression. Tumor stroma consists of a
heterogeneous population of cells, which include endothelial, immune, smooth muscle,
and most abundantly fibroblast cells, and hormones, growth factors, and cytokines [64].
In normal prostate, this mesh of cells, proteins, and hormones act together to keep tissue
homeostasis including growth and differentiation [25]. When the prostate epithelial cells
are transformed, altered cells then recruit or induce adjacent fibroblast cells to become
‘activated’ which in turn start the conversion of normal microenvironment into a tumor
associated one [68]. In many different types of cancer including prostate, the tumor

2
microenvironment has been shown to contribute to both the development of the primary
tumor and metastasis. In Paget’s ‘seed and soil’ hypothesis, the site of metastasis is
dependent on the microenvironment of the distant organ. Prostate cancer has a
propensity for bone metastasis, occurring in 65-75% of advanced cases [7,12] Therefore,
it is important to know what factors in the bone microenvironment leads to tumor
colonization as well as contribute to the osteoblastic nature of prostate bone metastasis.
CANCER ASSOCIATED FIBROBLASTS (CAFS)
The stromal microenvironment of the tumor has a major role in support of the growth,
survival, and dissemination of tumor cells. Most of the cells in the stroma are fibroblasts
and among them, the myofibroblasts or so-called “activated fibroblasts”. Activated
myofibroblasts express α-smooth muscle actin (α-SMA) which is most prominent in
prostate cancer [46, 45].
Fibroblasts are involved in all stages of cancer progression. They produce growth factors,
chemokines, and extracellular matrix proteins which results in angiogenesis and
recruitment of endothelial cells and pericytes. The enriched population of myofibroblasts
in the tumor stroma is associated with the production of increased levels of stromal cell–
derived factor-1 (SDF-1), also called CXCL12 ligand [7,8] Therefore, fibroblasts play a
major role in malignant progression of cancer and such present a good target for cancer
therapy. Fibroblasts are non-vascular, non-epithelial and non-inflammatory cells of the
connective tissue. Fibroblasts also secrete matrix metaloproteases (MMPs). By secreting
certain cytokines and growth factors they maintain the homeostasis of adjacent epithelial
cells and direct mesenchymal-epithelial interactions. In healthy organs they play major

3
role in wound healing, scar formation, and tissue fibrosis. They are the main component
of the connective tissue responsible for deposition of extracellular matrix (ECM), and
produce the fibrillar ECM such as type I, type III and type V collagen, and fibronectin.
They form the basement membrane by secreting type IV collagen and laminin [28].
DEVELOPMENT OF CELL LINES FROM ANDROGEN DEPENDENT
CANCER (AD-CA) AND CASTRATION-RESISTANT PROSTATE CANCER
(CRPC)
Androgen is the male sex steroid hormone which works through an androgen receptor
(AR). Androgen is needed for the differentiation and maturation of normal prostate as
well as the growth and proliferation of prostate cancer. In the early phase, the
development and progression of prostate cancer is still androgen-dependent (AD). [33].
However, over time, tumors become resistant to androgen deprivation, which results in a
more aggressive phenotype, castration-resistant prostate cancer (CRPC) [33]. A clear
understanding of the emergence of CRPC is yet to be manifested [33].
Two pairs of cell lines were generated in our lab from the conditional Pten deletion
mouse model [33]. The AD-Ca cell lines (E2, E4, and E8) and the ADI-Ca (CRPC) cell
lines (cE1 and cE2) display a similar level of expression of the androgen receptor (AR)
[33]. In the absence of androgen, the cE series grows well, displays increased AR
transcription, and retains the sensitivity to increased proliferation when androgen is
supplemented. The E series grows slowly in the absence of androgen, and does not show
increased AR [33]. The E series also produce very low levels of cytokeratins, but
increased levels of markers associated with epithelial-mesenchymal transition; however,
cE series produces a variety of epithelia cell markers [33]. Histopathological

4
examinations show that the cE series engenders adenocarcinomas, and the E series
induces sarcomatoid carcinomas [33]. These new cell lines should serve as useful
resources for understanding some of the molecular parameters contributing to the
progression and histopathology of prostate cancer [33].
BONE MORPHOGENETIC PROTEINS AND PROSTATE CANCER
Bone morphogenetic proteins (BMPs) belong to the transforming growth factor (TGF-β)
super-family. They are secreted extracellular signaling molecules first isolated from bone
matrix [49]. They are also involved in cell growth, differentiation, migration, and
apoptosis, and subsequently, they were shown to be imperative in development and
skeletal formation [49]. Like other members of the TGFβ family, BMP signaling is
initiated by the complexing of a type I and type II transmembrane serine/threonine
receptor with the BMP protein [17]. Activation of signaling pathways downstream of
receptor signaling consist of two categories: Smad-dependent and Smad-independent
pathways. In Smad-dependent signaling, phosphorylation of the serine/threonine sites on
the type I receptor leads to recruitment and phosphorylation of R-Smads (Smad 1, 5, and
8) [43]. Phospho-Smad 1, 5, and 8 are able to dimerize with Co-Smad (Smad 4) and this
complex is able to translocate to the nucleus [43]. Not much is known about Smad
independent pathways although it is established that TGFβ Activated Kinase 1 (TAK1)
activation by association with BMPRIA initiates these pathways[56].
Action of BMP in prostate cancer is known to be diverse, variable, and frequently cell
context and experimental context dependent. For example, it has been found that BMP-2
inhibits the growth of the androgen-dependent prostate cancer cell line, LNCaP [27,63]

5
but in castration resistant PC3 cells, some findings reported BMP-2 stimulated migration,
invasion, as well as proliferation [19, 31] while others reported the opposite [2]. BMP-4
was also able to stimulate migration and invasion in PC3 [19], but not in LNCaP or C42B
cell lines. BMP-6 was shown to inhibit growth in DU145 cells [24], but another report
shows that it had no effect on proliferation and instead enhanced migration and invasion
in PC-3 and DU145 cells [16]. BMP-7 was found to have an anti-proliferative effect on
LNCaP, PC3, and DU145 [54,8]. There is evidence that BMP-9 and -10 may have a
tumor suppressor role by inducing apoptosis in PC3 cells as well as slowing migration
and invasion [74,5].
STROMAL DERIVED FACTOR-1 (SDF-1)
SDF-1 is a cytokine expressed by stromal cells of different tissues such as endothelial
cells, pericytes, dendritic cells, fibroblasts, vascular smooth muscle cells from the skin,
osteoblasts and endothelial cells from bone marrow, and neurons from the brain [47, 48,
51, 62]. There are two SDF-1 isoforms, SDF-1α and SDF-1β, in human and mouse.
These two isoforms arise from a single gene through alternative splicing [46]. The only
difference is that SDF-1β has a four amino acid COOH-terminal extension [46]. SDF-1
is very conserved between species. Human and mouse SDF-1α and SDF-1β are 99% and
97% identical in amino acid sequences, respectively [46]. No differences have been
reported between SDF-1α– and SDF-1β–regulated expression and biological activity
[46].
It has been shown that CXCR4 is among the genes, which is expressed in high levels in
metastatic stages of prostate cancer, therefore, CXCR4 is a potential target for therapeutic

6
intervention in malignancies that metastasize [10]. As shown in figures 10 and 11,
SDF-1 specifically binds to CXCR4, which results in activation of tumor progression
pathways, such as tumor proliferation, angiogenesis, and invasiveness. These pathways
included: G-protein-coupled receptor (GPCR) signaling; PI3K/AKT; MAPK;
JAK/STAT; Src kinase; and HER2 [10]. Also, binding of SDF-1 to CXCR4 leads to
expression and secretion of MMP-9 through both the PI3 kinase and MAP kinase
pathways [11]. MMP-9 production and cell migration is also AKT dependent [11].
Interestingly phosphatase and tensin homolog (PTEN), which functions as a dual
specificity lipid and protein phosphatase, inhibits tumorigenic events through PI3K/AKT
[10]. Loss of PTEN expression in prostate cancer cells results in loss of a critical
inhibitory role of this protein, which leads to cell migration, proliferation, and invasion
[10]. It has been shown that PTEN and CXCR4 follow the same pathway of PI3K/AKT
and/or ERK1/2 signaling during tumor progression, and that loss of PTEN expression
results in loss of a critical inhibitory function in the signal cascade, permitting CXCR4-
mediated cell migration, proliferation, and invasion [10].
Particular attention has been paid to the SDF-1-CXCR4 axis in prostate cancer bone
metastasis, as SDF-1 is able to significantly increase the adhesion of human prostate
cancer cells to the osteoblast and endothelial cell culture models, and to enhance their
migration and invasion [61]. In vivo, administration of a CXCR4-neutralizing antibody
or blocking peptides significantly reduces the ability of PC-3 prostate cancer cells to
metastasize and grow in bone [59].

7
HYPOTHESIS AND RATIONALE
The role of BMPs and SDF-1s in prostate cancer is a topic of much interest. The
survival rate for a patient diagnosed with prostate cancer is much lower after metastasis.
Since prostate tumor cells have a clear propensity for bone metastasis, there has been
great interest in studying the interactions between tumor cells and the bone
microenvironment. It is believed that both bone morphogenetic proteins and stromal cell
derived factor-1 play a role in bone metastasis. It has been shown that SDF-1 is able to
significantly increase the adhesion of human prostate cancer cells to the osteoblast and
endothelial cell culture models, and to enhance their migration and invasion. It is
believed that bone morphogenetic proteins (BMPs) in the bone microenvironment may
promote cancer cell colonization and survival. Also BMPs in tumor cells may contribute
to tumor progression, metastasis, as well as promote osteomimetic properties in the cell
allowing them to thrive in the bone microenvironment.
In our lab there have been several observations on the effect of BMPs on tumor cells.
Using our Pten null prostate tumor mouse model, it has been found that BMP-2 and
BMP-7 are consistently up-regulated with the growth of the tumors. To determine the
relevance of this observation to human prostate cancer, we found that several BMPs and
their receptors, type I and type II, are expressed in a number of different human prostate
non-neoplastic cell lines, cancer cell lines, and stromal cell lines [69]. Also, previous
research in our lab has shown that the cancer associated fibroblast (CAF) cells express
type I and type II BMP receptors as well as the receptor for SDF-1, CXCR4 [69]. SDF-1
activation is associated with BMP-induced Smad phosphorylation, and the stimulatory

8
effect is blocked by BMP antagonist, noggin [69]. The findings that BMP treatment can
increase SDF-1 pre-mRNA levels in a time-dependent manner and actinomycin D
treatment can abolish stimulatory effect of BMP suggest a transcriptional modulation of
SDF-1 by BMP signaling [69].
The focus of this dissertation is to extend the study of BMP-2 and SDF-1 using
homologous prostate cancer cells and cancer-associated fibroblasts to determine the
effect of those secreted factors on the property of these cellular compartments. Our
overall hypothesis is that the tumorigenic role of BMP-2 and SDF-1 in prostate cancer
may be regulated by both the context of the prostate cancer, e.g., AD-cancer or CRPC,
and the source of the cancer-associated fibroblasts, e.g. AD-CAF or CRPC-CAF.

9
MATERIALS AND METHODS
CELL CULTURES
All cell cultures used for this project were obtained from the conditional Pten
deletion/luciferase reporter activation (cPten
-/-
L ) mice prostate cancer tissues as
described previously [69]. The AD-CAF primary cultures (No. 481, 3494, 2160, 3926,
3270), and one available CRPC-CAF primary culture (No. 3266) used for all the
experiments were within 7-10 passages. I did not let the cells to go over passage 10. All
the CAF cells were plated in medium containing Dulbecco’s Modified Eagle Medium
(DMEM) (Invitrogen) supplemented with 5% fetal bovine serum (FBS), 5% Nu serum
(BD Biosciences), 5.0 μg/ml insulin(Sigma-Aldrich), and 1% penicillin/streptomycine
(P/S). The NIH 3T3 cells were plated in DMEM (Invitrogen) supplemented with 10%
fetal calf serum (FCS), and 1% P/S. The prostate cancer epithelial cells were plated in
RPMI 1640 supplemented with 10% FBS, 1% P/S, and 5.0 μg/ml insulin. All the cells
were incubated in a humidified atmosphere of 5% CO
2
at 37°C. When the stromal cells
had reached 70–80% confluency, they were detached using 0.25% trypsin/1 mm EDTA
and re-plated at 20% densities.
LUCIFERASE ASSAY
To determine the biological activity status of BMP-2 molecules used in the experiments,
we used a reporter assay to confirm bioactivity. MC3T3-E1 cell cultures transfected with
the Bmp2-Smad reporter, (GCCG)
12
-luciferase, reporter constructs, and also the new
BMP-2 protein stock, were both kind gifts from Dr. Baruch Frenkel, Institute for Genetic

10
Medicine, Keck School of Medicine, Los Angeles, CA. MC3T3-E1 cell cultures were
plated in 6 well plates and maintained in Dulbecco’s Modified Eagle Medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) and 1.5% penicillin/streptomycine.
Starting at 90% confluency, the culture medium was supplemented with 200ng/ml of
BMP-2 overnight. The next day cell lysates were prepared in a 1X lysis buffer. 100 μl
of Luciferase Assay Reagent was dispensed into luminometer tubes, one tube per sample.
The Perkin-Elmer VICTOR luminometer (Perkin-Elmer Life Sciences) was programmed
to perform a 2 second measurement delay followed by a 10 second measurement to read
for luciferase activity. 20 μl of cell lysate was added to 100 μl of luminometer tube
containing the luciferase assay reagent, mixed by pipeting 2-3 times or vortex briefly.
The tube was then placed in the luminometer to initiate reading.
RNA PREPARATION AND QUANTITATIVE REAL-TIME-PCR (QRT-PCR)
Total RNA was extracted from E8, cE1, and NIH 3T3 cell cultures, by TRIzol reagent
(Invitrogen, Inc.) after the protocols recommended by the manufacturers. DNaseI
(Ambion) was used to remove contaminants of genomic DNA in the RNA samples. The
RNA (2 μg) was reverse transcribed by using iScript DNA Synthesis kit (Bio-Rad). The
synthesized cDNA was subjected to qRT-PCR with the primers described in Table 1.
The Real-time PCR was carried out using 13 μL of 2× Brilliant SYBER Green QPCR
Master Mix (Stratagene) with 1 μL of cDNA in a total volume of 25 μL. The PCR
conditions were as follows: 1 cycle of 95°C for 10 min; 40 cycles of 95°C for 30 s, 55°C
for 1 min, and 72°C for 30 s; and 1 cycle of 95°C for 1 min and 55°C for 30 s. Reactions
were carried out with the Stratagene Mx3000P PCR machine, and the cycle thresholds

11
were determined with its accompanying software. Actin was used for each sample as
control.

12

Table 1. Primer sequences for qRT-PCR

ELISA ASSAY FOR MEASURING SDF-1 SECRETION
Various AD-CAF (481, 3494, 2160, 3926, 3270) and CRPC-CAF 3266 cell cultures were
seeded at 350,000 cells/well in a 4 well plates, in the same Bfs medium that was used to
isolate them. After 24 hr. when the cells were 100% confluent, cell media was changed
from Bfs to serum free media (SFM) with 0.1% bovine serum albumin, 5μg/mL insulin,
and 0.5 μg/ml R1881. Then cells were treated with 2 μl of 100 ng/ml of the tested BMP-
2 protein stock. The final concentration of BMP-2 for this experiment was 200 ng/ml.
As control 2 μl of DMEM was used. After 24 hr. the conditioned medium from CAF
cells were collected and stored at -80 ˚C. The mouse SDF-1 concentration in the
conditioned medium from CAF was measured using a commercially available SDF-1
ELISA kit (R&D Systems). Because the number of the CAF cells and the volume of SFM
used for conditioned medium preparation varied in different experiments, the ELISA
results were further normalized by cell number and presented as total amount of SDF-1 α
secretion per one million cells (ng/million cells).

Gene Forward Primer Reverse Primer Source
CXCR4 ACG GCT GTA GAG CGA GTG TT AGG GTT CCT TGT TGG AGT CA IDT
78115774

13
PROLIFERATION ASSAY FOR TESTING SDF-1 α EFFECT ON PROSTATE
CANCER EPITHELIAL CELLS
The recombinant murine SDF-1 α was purchased from PeproTech, Inc. E8 and cE1 cells
(0.5 X 10
5
) were plated in each well of the six-well plate in triplicates in the absence or
presence of 300ng/mL of SDF-1α. Cells were counted using a Coulter counter
(Beckman Coulter, Inc., Miami, FL) every 2 days. The culture medium was changed and
SDF-1 α replenished every 2 days.
CELL CULTURE WOUND HEALING ASSAY FOR TESTING SDF-1 α EFFECT
ON PROSTATE CANCER EPITHELIAL CELLS
After E8 and cE1 cells were treated with 100 ng/ml SDF-1 α for 6 days and reached 90%
to 100% confluency, the wound line was made with a microtip on the monolayer cultures
of the cells. Cells were washed with PBS twice, fresh medium was added, and
photographs were taken at indicated time points.
STATISTICAL ANALYSIS
All the result of ELISA, qRT-PCR, proliferation assay, and migration assay were
evaluated as the mean ± SE of three independent repeats. Statistical calculations were
done with Microsoft Excel analysis tools. Differences between individual groups were
analyzed by independent t test. P value of <0.05 was considered statistically significant.

14
RESULTS
FUNCTIONAL VIABILITY OF OUR BMP-2 PROTEIN STOCK
To assess the bioactivity of our BMP-2 protein stock, I used the luciferase assay
technique. For this purpose I was given the MC3T3-E1 cell stock which was previously
transfected by the BMP-2-Smad reporter, (GCCG)
12
-luciferase, reporter construct.
While assessing the effectiveness and viability of our new BMP-2 protein stock on the
MC3T3-E1 cell cultures, I included as a positive control the old BMP-2 protein stock,
which has been staying for 2 months in the -80˚C freezer, and a vehicle control from
Dulbecco’s Modified Eagle Medium (DMEM). As expected when the new stock of
200 ng/ml of BMP-2 protein was used to treat the MC3T3-E1 cells for 24 hour, the
treatment resulted in robust stimulation of luciferase activity, validating responsiveness
of the reporter to BMP-2 signaling (Figure 1).
Luciferase Assay
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Vehicle Control Old BMP-2 (200
ng/ml)
New BMP-2 (200
ng/ml)
MC3T3-E1 cell cultures treated with BMP-2
Relative Luciferase Activity
Vehicle Control
Old BMP-2 (200 ng/ml)
New BMP-2 (200 ng/ml)
Figure 1. Potency of the lot of BMP-2 used in this work. Luciferase activity is
expressed relative to the mean. Data are presented as mean ± SD (n=3).

15
BMP-2 STIMULATES SDF-1 SECRETION IN AD-CAF AND CRPC-CAF
To test a possible relationship between BMP-2 and SDF-1α signaling in AD-CAF and
CRPC-CAF cell cultures, I measured SDF-1 secretion in our five AD-CAF primary
cultures (No. 481, 3494, 2160, 3926, and 3270), and one available CRPC-CAF primary
cultures (No. 3266), after treatment with BMP-2. It is found that BMP-2 up-regulates
SDF-1 secretion in all the CAF cell cultures, although to a variable degree; however, the
effect of BMP-2 is most strong, with 2-fold up-regulation observed at the 200 ng/ml
concentration, in CRPC-CAF cell cultures relative to AD-CAF cultures (Figure 2). Our
results indicate that SDF-1 secretion increases dramatically in CRPC-CAFs from the
recurrent prostate cancer tissue when exposed to extracellular BMP-2 (Figure 2).
However, it should be noted that for determining the validity of the finding, additional
CRPC-CAF cultures should be tested.

16

Figure 2. BMP-2 stimulates SDF-1α secretion in AD-CAF and CRPC-CAF cell
cultures. Conditioned medium (CM) from AD-CAF and CRPC-CAF cells after 24
hr. treatment with 200 ng/ml of BMP-2 were assayed for SDF-1 α concentration by
ELISA, and the level of secreted SDF-1α represented as nano-gram per one million
cells (ng/million cells), was compared among different groups by normalization with
conditioned medium volume and cell number. Data are presented as mean ± SD
(n=3). P<0.05.
NEW MOUSE PROSTATE CANCER EPITHELIAL CELLS EXPRESS CXCR4
Expression of SDF-1 receptor was investigated using quantitative reverse transcription-
PCR (qRT-PCR). As shown in Figure. 3 both prostate cancer epithelial cell cultures (E8
and cE1) were positive for the expression of CXCR4. NIH 3T3 cell line that doesn’t
express CXCR4 served as a negative control (Figure 3). It is noteworthy that the steady
state level of CXCR4 transcripts appears significantly higher, with 3-fold up-regulation,
in the E8 cells, representing the primary tumor cells, in comparison to cE1 cells, which
were derived from a CRPC tumor.

BMP-2 induction of SDF-1α
0
2
4
6
8
10
12
14
16
18
20
AD-CAF
3494
AD-CAF-
481
AD-CAF-
2160
AD-CAF-
3926
AD-CAF-
3270
CRPC
3266
CAF cell cultures
SD F-1α (ng/million of cells)
Vehicle Control (ng/million of cells)
BMP2 (ng/million of cells)
P=0.007

17

Figure 3. Detection of CXCR4 expression in two different types of prostate epithelia
cell cultures: E8 and cE1. Expression level of CXCR4 is normalized with a
expression of a known protein β-actin as a control in NIH 3T3, cE1 and E8 cell
cultures. Data are presented as mean ± SD (n=3). P<0.05.
SDF-1 INDUCES PROLIFERATION OF E8 and cE1
The effect of SDF-1 on the growth of E8 and cE1 prostate epithelial cells was tested.
Since E8 and cE1 cell cultures could sustain growth in medium with reduced amount of
serum (% 0.5) for a reasonable period of time, the serum source for the SDF-1 could be
largely avoided. A concentration of added SDF-1 (300 ng/ml) was selected that was
previously described to be optimal in concentrations for determining the effect on
mammalian cells in culture [34]. SDF-1 (300 ng/ml) did not affect the growth of cE1
cells compared to control. In contrast, the growth of E8 cells was significantly enhanced
up to 40% on day 5 of treatment (Figure 4). A reasonable explanation for this increased
sensitivity of E8 cells relative to the cE1 cells is perhaps due to the observed higher level
of CXCR4 expression in the E8 cells.
qRT- PCR
0
0.000005
0.00001
0.000015
0.00002
0.000025
0.00003
0.000035
0.00004
3T3 cE1 E8
Cell Cultures
Mean ratio to β-actin
3T3
cE1
E8
P=0.001

18

Figure 4. cE1 and E8 cell growth in medium containing 0.5% serum was
determined by counting the cell number every 2 days in the absence or presence of
300ng/mL of SDF-1 α recombinant protein. Data are presented as mean ± SD (n=3).
BMP-2 INDUCES EPITHELIAL-MESENCHYMAL TRANSDIFFERENTIATION
IN E8 AND cE1
We observed that exposure of both E8 and cE1 cells to exogenous BMP-2 results in a
remarkable morphologic change in these cells as shown in Figure 5. The cells treated
with 200 ng/ml BMP-2 for 24 hr. appear to lose their polygonal shape, a distinctive
characteristic of the epithelial cells, to acquire an elongated spindle-shaped morphology,
a phenotype more similar to fibroblastic cells. The E8 cells were previously reported to
display more of fibroblastic than epithelial characteristics [33]. Here, we observe that
shift could be further induced by BMP-2. In case of cE1 cells which are known to
maintain epithelial phenotype, exogenous BMP-2 appears to enhance the morphologic
Proliferation Assay
0
20
40
60
80
100
120
140
160
180
200
Day1 Day3 Day5
DAY
Cell Number (X10
3
)
E8 Vehicle control
E8 SDF-1α ( 300 ng/mL)
cE1 Vehicle control
cE1 SDF-1α( 300 ng/mL)

19
EMT shift, although bulk of the cells remains the epithelial phenotype.
Figure 5. Light microscopy pictures illustrate the morphology of cE1 and E8 cells
after treatment with 200ng/mL of BMP-2 for 24 hr.
SDF-1 INDUCES CELL MIGRATION OF E8 AND cE1
Prostate epithelial cell cultures E8 and cE1 cells were examined for their mobility in
culture plates. After E8 and cE1 cells were treated with 100 ng/ml SDF-1 for 6 days,
they were subjected to cell culture wound healing assay. As illustrated in Figure 6-8,
movement of E8 cells into the wound line was found to be clearly faster than the cE1
cells. By 15 hour after wounding SDF-1-treated E8 cells could move in to fill up the
space by ~50%, whereas only a few scattered cells were detected in the wound made in
cE1 cell cultures. Thus, it appears that E8 cells expressing higher levels of CXCR4
receptor than the cE1 cells, do respond better, whether it is proliferation or migration, to
supplemented SDF-1.
cE1

E8
No BMP-2 No BMP-2
BMP-2
(200 ng/ml)
BMP-2
(200 ng/ml)

20

Figure 6. Representative light microscopy pictures to indicate increased motility
into a wound line of E8 cells which were exposed to 100ng/mL of SDF-1α relative to
the control cells.

5hr
15hr
E8, 100 ng/ml SDF-1 α
0hr
5hr
15hr
0hr
E8, 0 ng/ml SDF-1 α

21

Figure 7. Representative light microscopy pictures to indicate increased motility
into a wound line of cE1 cells which were exposed to 100ng/mL of SDF-1α relative to
the control cells.

0hr
5hr
15hr
cE1, 0 ng/ml SDF-1 α cE1, 100ng/ml SDF-1 α
0hr
5hr
15hr

22

Figure 8. Migration rate of E8 and cE1 cells into the wound line in μm/hr. in the
presence of 100ng/ml of SDF-1α and absence of SDF-1 α. Migration rate of E8 cells
after treatment with 100ng/ml of SDF-1 α was highest compared to cE1 cells. Data
are presented as mean ± SD (n=3). P<0.05.

Quantitation of the Migration Rate in the Wound Healing Assay
0
0.2
0.4
0.6
0.8
1
1.2
cE1/Control cE1/SDF-1 α E8/Control E8/SDF-1 α
Epithelial Cell Cultures
M igration R ate (μm/hr)
cE1/Control cE1/SDF-1 α
E8/Control E8/SDF-1 α

23
DISCUSSION
It was reported earlier that the exogenous BMP-2 treatment of two different primary
fibroblastic cultures, each derived from a primary prostate tumor of the Pten null mouse
model, results in enhanced secretion of SDF-1, a chemokine that functions as both
chemoattractant for cell migration and mitogen for cell proliferation and survival [69]. In
the current study, we expanded the primary tumor or AD tumor CAF cultures by five
more in number to determine how consistent the observed effect of BMP-2 is. While
there is indeed a consistent enhancement of SDF-1 induction in all AD-CAF cultures, the
degree of induction appears to vary widely. It is noteworthy that the single CRPC-CAF
that we tested, however, appears to be distinct from the AD-CAFs in regard to two
parameters. First, the CRPC-CAFs are found in repeated experiments, to naturally
secrete a strong level of SDF-1, 4-fold higher than the best level seen in the AD-CAFs.
Second, the induction of SDF-1 by BMP-2 is similarly increased in the CRPC CAFs.
Thus it appears that the biological effect induced by BMP-2 is cell type dependent and
that fibroblast cells from castration resistant prostate cancer are more responsive to
extracellular BMP-2 and express higher amount of SDF-1. The SDF-1 induction by
BMP-2 that we found is not confined to only CAFs. A similar effect is observed in the
fibroblasts derived from normal prostate tissues [69]. These results suggest that
regardless of the origin of the prostate fibroblasts, and despite the variation of the
constitutive levels of SDF-1 in these cell populations, any increase in extracellular
BMP-2 is likely to positively induce SDF-1 expression in the prostate tissue
microenvironment.

24
Of the multiple mechanisms by which CAF may support tumor growth, secretion of
elevated levels of proteases, growth regulators, and extracellular matrix proteins have
been implicated to date [13]. In human breast and prostate cancer, several studies have
shown that SDF-1 is overexpressed in CAF and can contribute to both tumor growth and
angiogenesis [34, 13, 72]. Also, in this work, it is found that CXCR4 (SDF-1 receptor)
expression is significantly higher in epithelial cells of androgen dependent prostate
cancer (E8 cells) compared to those from castration resistant prostate cancer (cE1 cells).
Thus, it was important to evaluate the effect of SDF-1 on these cell cultures. When both
E8 and cE1 cells are treated with SDF-1, E8 cells are induced to proliferate at a much
higher rate compared to cE1 cells. SDF-1 also causes E8 cells to migrate to the wound
line at a much higher rate. This observation may be due to the fact that CXCR4, the
receptor for SDF-1 protein, expression is much higher in E8 cells compared to cE1 cells.
It has been shown that cE1 cell cultures produce a variety of epithelial cell markers [33].
The E8 cells produce very low levels of cytokeratins, but increased levels of markers
associated with epithelial-mesenchymal transition (EMT) [33].
There is also a new observation in this study that E8 and cE1 cells when treated with
BMP-2, can undergo epithelial mesenchymal transdifferentiation (EMT). This effect is
more pronounced on E8 cells than cE1 cells. Thus, extracellular BMP-2 not only induces
SDF-1 secretion in cancer associated fibroblasts, while at the same time appears to
promote EMT in prostate cancer epithelial cells. Treated epithelial cells appear to lose
their polygonal shape and adhesive cell contacts, which are distinctive of epithelial cells
and to acquire an elongated spindle-shaped morphology, a phenotype more similar to
fibroblastic cells. These transformed prostate epithelial cells (E8 cells) also express high

25
level of CXCR4 which is a receptor for SDF-1. Therefore, E8 cells representing primary
tumors could gain from recruiting activated cancer associated fibroblasts for growth.
SDF-1 expression will have then a pro-tumorigenic effect. As CXCR4 is found to be
present in the CAF, whether SDF-1 may also have autocrine effects on CAF is still
another point that remains to be defined.
Although based on the scrutiny of a single CRPC-CAF culture, the clues appear to
display a different scenario for the CRPC cancer and associated CAFs. cE1 cells
representing the recurrent prostate cancer produce only low levels of CXCR4 receptor
transcripts but the associated CAFs (CRPC-CAFs) secrete high levels of SDF-1 and
respond to BMP-2 strongly to produce more SDF-1. It seems that a balance in SDF-1-
CXCR4 interactions in CRPC is restored through compensation of reduction of receptor
expression by the cE1 cells relative to E8 cells by increased levels of SDF-1 in the
CRPC-CAF compartment. This exciting clue remains to be better developed in the
future.
Taken together, the evidence for induction of SDF-1 by BMP-2 in AD-CAF and
CRPC-CAF along with the demonstrated expression of CXCR4 in both prostate cancer
epithelial cells, E8 and cE1 cells, seem to indicate potentially important heterotypic cell-
cell interactions driven by both autocrine and paracrine mechanisms in prostate cancer.
These results further underscore the contention that the intervention of BMP-2 and SDF-1
signaling activity may lead to a potential therapeutic treatment for prostate cancer.
Further mechanistic studies are also in order. We need to know whether BMP-2 acts in a
autocrine or paracrine fashion in inducing SDF-1 secretion. To do so we can treat cells

26
with an inhibitory molecule of BMP-2 signaling pathway such as Noggin, and detect the
basal level of SDF-1 in the condition media. If the basal level of SDF-1 does not change
with Noggin treatment, we can conclude that endogenous functional level of BMP-2 in
AD-CAF and CRPC-CAF cells is sufficient to enhance SDF-1 expression. The second is
what kind of intracellular signaling events trigger SDF-1 secretion as a result of
extracellular BMP-2 treatment of AD-CAF and CRPC-CAF cell cultures. Previous
research in our lab has shown that BMP-2 induces Smad phosphorylation in primary
CAF (Figure 9B). When CAF cells are treated with 1 μg/ml of Noggin, in the presence
of 200 ng/ml of BMP-2, the ability of BMP-2 to cause Smad phosphorylation is
abolished (Figure 9B) [69]. Also in the presence of Noggin the stimulatory effect on
SDF-1 secretion was nullified and reduced (Figure 9A-9B) [69]. Therefore, SDF-1
secretion via BMP-2 is a Smad- dependent pathway in CAF cell cultures.
In short, this report sets-up a new oncogenic property of BMP-2-SDF-1 axis in two
different types of the prostate tumors, the primary and the recurrent prostate cancer,
introducing an interesting aspect of heterotypic cell interaction potentially critical in
prostate cancer.

27
A.

B.

Figure 9. BMP-2 stimulates SDF-1 secretion and induces Smad phosphorylation in
CAF-1 cell. (A) Conditioned medium (CM) from CAF-1 cells with different 24 h
treatments as indicated in the charts were assayed for SDF-1 concentration by
ELISA, and the level of secreted SDF-1, represented as nanogram per one million
cells (ng/million cells) normalized with conditioned medium volume and cell number.
(B) The same CAF cell that is used for ELISA was lysed and subjected to Western
blot analysis to detect Smad phosphorylation and the expression of Smad 5.
Analysis of actin was used as a loading control [69].

SDF-1 Secretion in CAF-1
0
10
20
30
40
CONT Noggin
1ug/ml
BMP2
100ng/ml
BMP2 +
Noggin
ng/million cells

28

Figure 10. Proposed mechanism for the role of PTEN in CXCR4-mediated
signaling. A, ligand activation of CXCR4 stimulates several pathways, including
PI3K/AKT and ERK1/2, resulting in tumorigenic events. PTEN negatively
regulates both pathways by acting as a protein and lipid phosphatase. B, loss of
PTEN expression results in a loss of regulation of CXCR4-mediated events,
permitting activation of signaling pathways that enhance tumorigenesis [10].

29

Figure 11. Schematic representation of CXCL12-induced CXCR4 activation in
cancer cells in the bone microenvironment. Exposure of PC cells to the bone
microenvironment leads to binding of the bone-derived CXCL12 chemokine to the
CXCR4 receptor in lipid rafts on the surface of cancer cells. This receptor-ligand
interaction activates both the PI3 kinase and the MAP kinase pathways leading
ultimately to Akt1 activation, NF-κB transcription factor activation, MMP-9 gene
expression, MMP-9 protein release, and cellular migration and invasion. These
findings link the process of chemoattraction to MMP9-mediated invasion, tumor
growth, and bone remodeling [11].

30
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Abstract (if available)

Abstract The focus of this study is to better understand the role of bone morphogenetic protein-2 (BMP-2), and stromal cell-derived factor-1 (SDF-1) that occur in the prostate cancer tumor microenvironment with respect to interactions between malignant epithelial cells and the associated fibroblastic cells. The research concerns two distinct phases of the disease: androgen-dependent primary tumor (AD cancer) and the post-castration androgen refractory tumor development-the castration resistant prostate cancer (CRPC). The model systems used are E8 (primary tumor), and cE1 (CRPC tumor) malignant epithelial cell lines, and primary cultures of cancer-associated fibroblasts, derived from primary tumor (AD-CAFs) or CRPC tumor (CRPC-CAFs). All cellular materials have a homologous origin from the conditional Pten deletion mouse model of prostate cancer. First, it is shown that all CAFs, AD or CRPC in origin, are responsive to BMP-2 induced stimulation of SDF-1 expression. While the level of induction varies from one AD-CAF to another, a single CRPC-CAF tested exhibits a robust induction of SDF-1 by BMP-2. Second, the constitutive level of secreted SDF-1 is significantly higher in CRPC-CAF as compared to AD-CAFs. In contrast to these observations on SDF-1 induction, the levels of the cognate receptor CXCR4 appears to be low in CRPC cancer cells (cE1) in relation to that in primary tumor cells (E8). This opposite spectrum of ligand and receptor expression may, perhaps, be a mode of operation to regulate tumor growth in the prostate tumor microenvironment. Third, the increased level of CXCR4 in E8 cells is demonstrated to correlate well with their responsiveness to SDF-1 for proliferation or migration potentials. Finally, it is possible that the high levels of SDF-1 produced by CRPC-CAFs may have other significant role, such as, in cancer cell invasion and tumor angiogenesis that are critical for the recurrent tumor growth and metastasis. These clues

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Creator Fata, Azadeh (author)

Core Title Study of bone morphogenetic protein-2 and stromal cell derived factor-1 in prostate cancer

School Keck School of Medicine

Degree Master of Science

Degree Program Pathobiology

Publication Date 05/06/2012

Defense Date 05/03/2012

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

Tag androgen-dependent primary tumor (AD cancer),bone morphogenetic protein-2,castration resistant prostate cancer (CRPC),malignant epithelial cells,malignant fibroblastic cells,OAI-PMH Harvest,prostate cancer,stromal cell derived factor-1,tumor microenvironment

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Kalra, Vijay K. (committee chair), Roy-Burman, Pradip (committee member), Widelitz, Randall B. (committee member)

Creator Email azfata@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-34271

Unique identifier UC11288786

Identifier usctheses-c3-34271 (legacy record id)

Legacy Identifier etd-FataAzadeh-791.pdf

Dmrecord 34271

Document Type Thesis

Rights Fata, Azadeh

Type texts

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

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

Repository Name University of Southern California Digital Library

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