Loss of PDEF, a prostate-derived Ets factor is associated with aggressive phenotype of prostate cancer: regulation of MMP 9 by PDEF.

Abstract

Prostate-derived Ets factor (PDEF) is expressed in tissues of high epithelial content including prostate, although its precise function has not been fully established. Conventional therapies produce a high rate of cure for patients with localized prostate cancer, but there is, at present, no effective treatment for intervention in metastatic prostate cancer. These facts underline the need to develop new approaches for early diagnosis of aggressive prostate cancer patients, and mechanism based anti-metastasis therapies that will improve the outlook for hormone-refractory prostate cancer. In this study we evaluated role of prostate-derived Ets factor (PDEF) in prostate cancer.
We observed decreased PDEF expression in prostate cancer cell lines correlated with increased aggressive phenotype, and complete loss of PDEF protein in metastatic prostate cancer cell lines. Loss of PDEF expression was confirmed in high Gleason Grade prostate cancer samples by immuno-histochemical methods. Reintroduction of PDEF profoundly affected cell behavior leading to less invasive phenotypes in three dimensional cultures. In addition, PDEF expressing cells had altered cell morphology, decreased FAK phosphorylation and decreased colony formation, cell migration, and cellular invasiveness. In contrast PDEF knockdown resulted in increased migration and invasion as well as clonogenic activity. Our results also demonstrated that PDEF downregulated MMP9 promoter activity, suppressed MMP9 mRNA expression, and resulted in loss of MMP9 activity in prostate cancer cells. These results suggested that loss of PDEF might be associated with increased MMP9 expression and activity in aggressive prostate cancer. To confirm results we investigated MMP9 expression in clinical samples of prostate cancer. Results of these studies show increased MMP9 expression correlated with advanced Gleason grade. Taken together our results demonstrate decreased PDEF expression and increased MMP9 expression during the transition to aggressive prostate cancer.
These studies demonstrate for the first time negative regulation of MMP9 expression by PDEF, and that PDEF expression was lost in aggressive prostate cancer and was inversely associated with MMP9 expression in clinical samples of prostate cancer. Based on these exciting results, we propose that loss of PDEF along with increased MMP9 expression should serve as novel markers for early detection of aggressive prostate cancer.

Full text

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Johnson et al. Molecular Cancer 2010, 9:148
http://www.molecular-cancer.com/content/9/1/148
Open Access
RESEARCH
Loss of PDEF, a prostate-derived Ets factor is
associated with aggressive phenotype of prostate
cancer: Regulation of MMP 9 by PDEF
© 2010 Johnson et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Research
Thomas R Johnson, Sweaty Koul, Binod Kumar, Lakshmipathi Khandrika, Sarah Venezia, Paul D Maroni,
Randall B Meacham and Hari K Koul*
Abstract
Background: Prostate-derived Ets factor (PDEF) is expressed in tissues of high epithelial content including prostate,
although its precise function has not been fully established. Conventional therapies produce a high rate of cure for
patients with localized prostate cancer, but there is, at present, no effective treatment for intervention in metastatic
prostate cancer. These facts underline the need to develop new approaches for early diagnosis of aggressive prostate
cancer patients, and mechanism based anti-metastasis therapies that will improve the outlook for hormone-refractory
prostate cancer. In this study we evaluated role of prostate-derived Ets factor (PDEF) in prostate cancer.
Results: We observed decreased PDEF expression in prostate cancer cell lines correlated with increased aggressive
phenotype, and complete loss of PDEF protein in metastatic prostate cancer cell lines. Loss of PDEF expression was
confirmed in high Gleason Grade prostate cancer samples by immuno-histochemical methods. Reintroduction of
PDEF profoundly affected cell behavior leading to less invasive phenotypes in three dimensional cultures. In addition,
PDEF expressing cells had altered cell morphology, decreased FAK phosphorylation and decreased colony formation,
cell migration, and cellular invasiveness. In contrast PDEF knockdown resulted in increased migration and invasion as
well as clonogenic activity. Our results also demonstrated that PDEF downregulated MMP9 promoter activity,
suppressed MMP9 mRNA expression, and resulted in loss of MMP9 activity in prostate cancer cells. These results
suggested that loss of PDEF might be associated with increased MMP9 expression and activity in aggressive prostate
cancer. To confirm results we investigated MMP9 expression in clinical samples of prostate cancer. Results of these
studies show increased MMP9 expression correlated with advanced Gleason grade. Taken together our results
demonstrate decreased PDEF expression and increased MMP9 expression during the transition to aggressive prostate
cancer.
Conclusions: These studies demonstrate for the first time negative regulation of MMP9 expression by PDEF, and that
PDEF expression was lost in aggressive prostate cancer and was inversely associated with MMP9 expression in clinical
samples of prostate cancer. Based on these exciting results, we propose that loss of PDEF along with increased MMP9
expression should serve as novel markers for early detection of aggressive prostate cancer.
Background
Prostate cancer is the second leading cause of cancer
death in men. In the United States alone, 192,280 new
cases of prostate cancers were diagnosed in 2009 and
among them around 27,360 deaths occurred. One of the
biggest challenges we face in prostate cancer is determin-
ing if the cancer is aggressive. Conventional therapies
produce a high rate of cure for patients with localized
prostate cancer, but there is no cure once the disease has
spread beyond the prostate. Reduction in serum prostate-
specific antigen (PSA) levels has been proposed as an
endpoint biomarker for human prostate cancer interven-
tion. However, despite being the mainstay of prostate
cancer detection, the value of PSA screening is still
* Correspondence: hari.koul@ucdenver.edu
1 Program in Urosciences, Division of Urology-Department of Surgery,
University of Colorado Denver School of Medicine, Denver Veterans
Administrative Medical Center, and University of Colorado Comprehensive
Cancer Center, Building P15 or RC2, C-317, 12700 E 19th Avenue, Aurora, CO
80045, USA
Full list of author information is available at the end of the article

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debated. In particular, there is a growing concern regard-
ing the over diagnosis of potentially indolent disease [1].
Therefore, there remains an urgent need for more accu-
rate biomarkers to diagnose aggressive prostate cancer.
Thus, identification of new molecular markers/targets for
aggressive prostate cancer is important in order to
improve early detection of the aggressive disease and to
develop new therapeutic regimens.
Progression of prostate cancer from focal, androgen-
dependent lesions to androgen-independent, metastatic
cancer requires deregulation of growth control, invasive-
ness and cell motility. Abundant evidence demonstrates
roles for Ets transcription factors in many cancers includ-
ing prostate. Prostate-derived Ets factor (PDEF), first
described nine years ago as preferentially binding to the
noncanonical Ets core sequence GGAT [2], has recently
received considerable attention due to its potential
importance in regulating cell motility and invasion [3-5].
Recently, proteomic analysis of PDEF overexpressing cells
revealed 286 proteins in the PDEF-associated protein
complex in breast cancer [6]. Thus interaction of PDEF
with other partner proteins could help in finding their
role in maintenance of malignant phenotype. Published
literature concerning experimental manipulation of
PDEF expression is paradoxical and limited to tissues of
high epithelial content, notably prostate, breast, ovary
and colon [7,8]. PDEF expression has been both positively
[3,9] and negatively [10] correlated with breast cancer
grade at mRNA or protein levels. It is important to note
that PDEF mRNA and protein levels do not always corre-
late, which may have led to different conclusions in some
of the studies examining PDEF expression in primary
tumors. Turner et al. [4] found that introducing PDEF
into invasive breast cancer cell lines reduced their invad-
ing ability. Similarly, siRNA-mediated knockdown of
PDEF in MCF7 cells increased their ability to migrate in
the Transwell assay. Besides its role in cancer metastasis,
PDEF expression was also correlated with changes in the
actin cytoskeleton and focal adhesion localization, and
loss of cellular polarity. Ghadersohi et al. [10] silenced
PDEF expression in MCF7 cells, and found that such cells
showed greatly accelerated xenograft tumor formation in
SCID mice. By contrast, Gunawardane et al. [3] showed
that increasing expression of PDEF increased their ability
to migrate in a Transwell assay and stimulated colony for-
mation in soft agar. This group also identified a canonical
MAP kinase phosphorylation site at T50 (PAT50P) and
showed that mutation to alanine at this site abolished all
the effects they observed. To date there are few data avail-
able formally correlating PDEF expression in mainte-
nance of prostate malignant phenotype. Two published
studies, one with a prostate cancer cell-line [5] and
another with clinical samples from prostate [11] reached
opposite conclusions with respect to the role of PDEF in
prostate cancer. Clearly additional studies are necessary
to evaluate role of PDEF in prostate cancer biology.
In the current studies, we report here that PDEF
expression is lost, whereas MMP9 expression increased
with the aggressive behavior of prostate cancer. Overex-
pression of PDEF in PC3 cells strongly inhibits colony
formation, cell migration and invasion, and increased cell
adherence. Furthermore, re-introduction of PDEF in PC3
cells led to changes in actin cytoskeleton, altered focal
adhesion kinase activity, and reestablished cell polarity in
these cultures as indicated by induction of less invasive
spheroid-like structures in three-dimensional culture,
Moreover, PDEF expression downregulates MMP9
expression, and its promoter activity in PC3 cells. Thus,
consideration of both PDEF and MMP9 may have a better
prognosis value for determining the aggressive phenotype
of prostate cancer.
Materials and methods
Constructs and cell lines
All cell lines (PC3, LNCaP, and C4-2B) were purchased
from ATCC and maintained according to ATCC guide-
lines. Phoenix cells were grown in DMEM containing
10% fetal bovine serum. FLAG tag antibody was pur-
chased from Sigma (St. Louis, USA). PDEF and phospho
FAK antibodies were from Santa Cruz Biotechnology
(CA, USA). PDEF was cloned from PC3 cDNA with an
amino-terminal FLAG tag, and inserted into retroviral
vectors pBABE and the bicistronic vector QCXIX (Clon-
tech). The latter vector was modified to contain a wild-
type internal ribosome entry site to increase expression
from the second multiple cloning site, into which G418
resistance was cloned. Mutations were created using the
Quick-Change kit (Stratagene) according to the manufac-
turer's instructions. Oligonucleotide primers for PCR
were purchased from Integrated DNA Technologies.
Retrovirus production and infection
Phoenix cells were transfected with 2 μg DNA using
Effectene (Qiagen) according to the manufacturer's
instructions, and infection was followed according to
Phoenix™ Retrovirus Expression System (Orbigen Inc.).
After infection, cells were trypsinized, transferred to 150
mm dishes, and subjected to puromycin or G418 selec-
tion after 48 h incubation.
Thymidine incorporation
1 × 105 cells/well were plated in 12-well plates. 48 h later,
cells were exposed to medium containing1-3 μCi/ml 3H-
thymidine (Perkin Elmer) and incubated for 4 h. After 2
washes with cold PBS, cells were fixed in cold methanol
for 5 min followed by an additional methanol wash. Cells
were solubilized in 0.1% SDS/0.2 M NaOH and radioac-
tivity determined.

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Anchorage independent growth, motility, invasion and
attachment assays
Growth in soft agar was performed as described previ-
ously [12]. Invasiveness was determined by the method of
Repesh LA [13]. Cell migration through Transwell mem-
branes was performed identically, but without the use of
Matrigel. Wound healing assays were performed by mak-
ing a cruciform scratch in a confluent monolayer of cells.
Cells were washed, the medium replaced with serum-free
medium, and incubated for 48-72 h. Cells were fixed with
methanol, stained with Giemsa, and photographed.
Attachment assays were performed essentially by the
method of Turner et al. [4] using 96-well plates pretreated
overnight with fibronectin, Matrigel, or bovine serum
albumin at concentrations of 50 ng/ml, 100 ng/ml, and 10
mg/ml respectively.
Immunohistochemistry for PDEF and MMP9 expression on
prostate tissue array slides
Tissue microarray slides containing 9 normal and 40
prostate cancer samples of varying pathological grade
were obtained from Imgenex Corporation, San Diego,
CA, 92121. Immuno histochemistry for PDEF and MMP9
was performed using the avidin-biotin complex method
previously described by Hsu et al. [14]. Expression of
PDEF and MMP9 were evaluated by analysis of micro-
scopic scans of each tissue. Expression was considered
high if greater than 60% of the scanned area scored posi-
tive, while expression was considered moderate if 40-60%
area scored positive, and expression was considered low if
less than 40% area scored positive.
Reverse transcription PCR
Total RNA was extracted using RNEasy mini kit (Qiagen).
1 μg of RNA was used to prepare cDNA using iScript sec-
ond strand cDNA synthesis kit (Bio-Rad). 100 ng of syn-
thesized cDNA was used for RT-PCR using forward 5-
TTGACAGCGACAAGAAGTGG-3 and reverse 5-
TCACGTCGTCCTTATGCAAG-3 for MMP9, forward
5-ACCACAGTCCATGCCATCAC-3 and reverse 5-
TCCACCACCCTGTTGCTGTA-3 for GAPDH. Tran-
scripts were separated by agarose gel electrophoresis.
Reporter assay
Cells were transfected with 1 μg of MMP9 luciferase
reporter vector along with 10 ng of Renilla luciferase
expression plasmid using Effectene (Qiagen, Valencia,
CA) according to manufacturer's instructions. Luciferase
activity was measured using the Dual luciferase kit (Pro-
mega Corporation, Madison, WI) with Monolight 2010
Luminometer (Analytical Luminescence laboratory, San
Diego, CA).
MMP Zymography
Zymogram for MMP9 activity was performed according
to Bernhard and Muschel [15] using conditioned
medium.
Three dimensional cell culture
PDEF overexpressing PC3 cells and respective vector
control cells were grown in growth factor reduced Matri-
gel for 10-12 days and then immunofluorescence staining
was performed according to Debnath et al. [16]
Morphology studies
Cell morphology was done on glass chamber slides by
immunofluorescence method. Vector control and PDEF
expressing cells were seeded on multi-chamber slide,
fixed with 4% formalin, permeabilized with 0.1% Triton-
X-100, blocked in 2% BSA, and change in actin cytoskele-
ton was examined by phalloidin staining as per the manu-
facturer's instructions (Molecular Probes, Eugene, OR).
Pictures were taken using Spin Disc Olympus confocal
microscope.
Western Blot analysis
Electrophoresis and blotting were performed as
described previously [17].
Statistical analysis
Statistical analyses for tissue culture studies were per-
formed using two-dimensional two sample variance T-
tests; For data from clinical specimens, statistical analysis
was performed using MANN - WHITNEY U: exact test.
p ≤ 0.05 was considered significant.
Results
PDEF expression is reduced during the transition from low
grade to high grade prostate cancer
PDEF expression was evaluated by immunohistochemical
examination in tissue microarray slides containing 40
cores of prostate cancer and 9 cores of normal prostate.
Results presented in Figure 1 show that PDEF protein
expression is downregulated during the transition to
aggressive prostate cancer. As shown in Figure. 1A, high
levels of PDEF protein are present in normal prostate epi-
thelial cells as well as early stage prostate cancer. How-
ever, in high grade prostate cancer PDEF protein is
significantly decreased. Significant reduction in PDEF
expression was observed in all cores of prostate tumor
tissue with high Gleason grade (Gleason score greater
than 7) as compared to normal prostate tissue as well as
low grade prostate cancer (Gleason score of 7 or below).
Moreover, PDEF protein levels showed graded decrease
with increase in pathologically confirmed aggressive dis-
ease. Our results show that while 59 ± 3.6% tissue scored
positive for PDEF in low to moderate grade (Gleason 6 &
7) prostate cancer, and 33 ± 3.3% of the tissue scored pos-
itive for PDEF in moderately high grade (Gleason 8)
tumor, there was little or no expression of PDEF in very
high grade (Gleason 9 & 10) prostate cancer(Figure. 1B).
Antibody specificity for these assays was determined by
Immunofluorescence and Western blot analysis using
cells with and without PDEF expression (Figure S1, Addi-
tional file 1). In addition to clinical samples, we also eval-

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uated PDEF protein expression in established prostate
cancer cell lines with low to high aggressive behavior.
These results presented in Figure 1C show that PDEF is
expressed in LNCaP cells (a less aggressive prostate can-
cer cell line). However, PDEF expression is reduced in
more aggressive lineages of LNCaP cells (C4-2 and C4-
2B). Moreover, PDEF expression is completely lost in two
widely used aggressive prostate cancer cells (DU145 and
PC3 cells). Taken together, results of these studies dem-
onstrate that PDEF expression is decreased or lost in
prostate cancer cells with aggressive phenotype, and pro-
vide novel insights into the characteristics of PDEF pro-
tein expression in progression of prostate cancers.
Absence of PDEF protein expression in PC3 cells in our
studies is in apparent disagreement with previous report
[5] that showed PDEF expression in PC3 cells,. This dis-
crepancy could result from several causes: First, the anti-
body used in [5] could be more sensitive, such that they
Figure 1 PDEF protein expression in human prostate tissues and prostate cancer cell lines. A, Representative photo-micrographs of Immuno-
histochemical analysis of PDEF expression using prostate tissue micro-array slides (containing both normal and tumor samples of different grades)
performed as described in Materials and Methods. B, Quantification of percentage staining for PDEF. C, Representative image showing Western blot
analysis on prostate cancer cell lines using anti-PDEF antibody (left panel), and quantitation of the same data (right panel).

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were able to detect even negligible amounts of PDEF. Sec-
ond PC3 cells change over various passages of culture and
media conditions, which could explain the differences.
Re-introduction of PDEF inhibited directional migration,
decreased cell migration and anchorage independent
growth in prostate cancer cells
To examine the effects of PDEF expression on cell motil-
ity, PC3 cells transduced with PDEF or mutant PDEF
T50A, or vector alone were assayed for their ability to
migrate through Transwell membranes as described in
materials and methods. Results presented in Figure 2A
indicate that both wild-type and mutant PDEF-trans-
duced cells were significantly inhibited in their ability to
migrate through Transwell pores. We also subjected cells
to an assay for persistence migratory directionality (in
vitro wound healing, Figure. 2B). Expression of either
PDEF or the T50A mutant inhibited the ability of PC3
cells to fill in gaps in a monolayer compared to vector
alone. These results demonstrated that PDEF signifi-
cantly interfered with ability of cells to maintain migra-
tory phenotype.
We next examined the effects of PDEF overexpression
on the ability of PC3 cells to form colonies in soft agar.
Expression of either PDEF or the T50A mutant equally
inhibited the ability of PC3 cells to form colonies in soft
agar (Figure. 2C) compared to vector alone.
To address the possibility that altered cell proliferation
contributed to the results of these assays, we measured
DNA synthesis (3H-thymidine incorporation) in control
and PDEF transfected PC3. Results of these studies (Fig-
ure. 2D) show that decreased clonogenic activity follow-
ing PDEF expression was not a consequence of decreased
DNA synthesis. These results demonstrated that PDEF
expression decreased clonogenic ability of the cells inde-
pendent of DNA synthesis. Moreover, in sharp contrast
to the effects of PDEF on anchorage independent growth,
PDEF expression did not significantly affect anchorage
dependent growth of prostate cancer cells in culture (data
not shown).
PDEF expression resulted in increased cell adhesion,
altered cell morphology and decreased focal adhesion
kinase activity in prostate cancer cells
Immunofluorescence studies of PDEF expressing cells
showed a rounded area of cleared fluorescence rather
than elongated track as seen on invasive vector control
cells (Figure. 3A). These results indicated that PDEF
expression resulted in alterations to actin cytoskeleton
and altered cell morphology. FAK is a non-receptor pro-
tein tyrosine kinase, associated with supramolecular focal
adhesion complexes. Focal adhesion complex assembly
and disassembly are critical for cell attachment and
movement [18]. The lack of morphologic polarity in
PDEF expressing cells as shown in Figure 3A raised the
possibility that PDEF may affect adhesion complex for-
mation. Moreover, in previous studies we observed that
FAK was non-phosphorylated in adherent cultures and
FAK phosphorylation was increased in suspension cul-
ture [19]. Therefore, we evaluated the effects of re-intro-
duction of PDEF in PC3 cells on FAK phosphorylation in
PC3 cells in suspension cultures. Results of these studies
revealed a significant reduction in FAK phosphorylation
in PDEF expressing cells grown in suspension culture
(Figure. 3B). These results demonstrated that PDEF
expression in PC3 cells resulted in decreased FAK activ-
ity, suggesting decreased focal adhesion formation.
Focal adhesion formation and its interaction with the
ECM play a central role in migration and invasion, since
increased adhesion makes cells less motile. To examine
this possibility, we directly measured the effects of PDEF
expression on adhesion of PC3 cells to various ECM sub-
strates. For these studies, PC3 cells transfected with
PDEF or vector alone were assayed for their ability to
attach to fibronectin or Matrigel-coated plastic surfaces.
Results presented in Figure 3C indicate that attachment
of PDEF-expressing cells to fibronectin-coated, Matrigel-
coated, or control (BSA treated) plastic was significantly
increased compared to vector-transduced cells. These
results are in contrast to the effects of PDEF in breast
cancer cells, where PDEF was shown to decrease adhe-
sion of the cells to fibronectin and matrigel [4]. Taken
together, these results suggest that PDEF mediated inhi-
bition of migration may occur through cytoskeleton dis-
organization and ECM interaction.
PDEF decreased invasion and inhibited expression of
matrix metalloproteinase-9 (MMP9) in prostate cancer cells
To test the effects of PDEF on cell invasion, we examined
the effects of PDEF expression on the ability of PC3 cells
to invade simulated basement membrane in vitro, a phe-
notype correlated with aggressive behavior. Results pre-
sented in Figure 4A indicate that expression of either
PDEF or the T50A mutant inhibited the ability of PC3
cells to invade through Matrigel compared with vector
transfected control cells. In addition to transfection of
PC3 cells with PDEF, we also performed complementary
RNA interference (RNAi) experiments to reduce the
endogenous PDEF expression in prostate cancer cells that
express PDEF (LNCaP and C4-2B cells), and directly
evaluated the effects of decreased PDEF levels on inva-
sion and clonogenic activity of these cells. Results pre-
sented in Figure S2, Additional file 1 demonstrated that
SiRNA mediated knock-down of PDEF in these cells
resulted in an increased ability to form colonies in soft
agar and increased invasion through Matrigel basement
membrane. Taken together with rest of the results these