Nuclear Factor-nB/p65 (Rel A) Is Constitutively Activated inHuman Prostate Adenocarcinoma and Correlates withDisease Progression1,2
Sanjeev Shukla*, Gregory T. MacLennan y,z, Pingfu Fu z,§, Jigar Patel y, Susan R. Marengo*, Martin I. Resnick*,z
and Sanjay Gupta*,z
Departments of *Urology and yPathology, Case Western Reserve University and University Hospitals of Cleveland,Cleveland, OH, USA; z Ireland Cancer Center, Cleveland, OH 44106, USA; §Department of Epidemiology andBiostatistics, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, OH, USA
Abstract
Aberrant nuclear factor-KB (NF-KB) activation has been
implicated in the pathogenesis of several human
malignancies. In this study, we determined whether
NF-KB is constitutively activated in human prostate
adenocarcinoma, and, if so, whether increased NF-KB
activation and its binding to DNA influence tumor
progression. Using tissue samples obtained during
transurethral prostatic resection and paraffin-embed-
ded sections of benign and cancer specimens, we
determined the nuclear expression of NF-KB/p65 and
NF-KB/p50, cytoplasmic expression of IKBA, its phos-
phorylation, and expression of NF-KB –regulated
genes, specifically Bcl2, cyclin D1, matrix metallopro-
teinase-9 (MMP-9), and vascular endothelial growth
factor (VEGF). A progressive increase in the expres-
sion of NF-nB/p65 (but not of p50) was observed in
cancer specimens compared to benign tissue, which
correlated with increasing levels of InBa and its phos-
phorylation. NF-nB DNA-binding activity increased with
increasing tumor grade and the binding complex
mainly consisted of NF-nB/p65–p50 heterodimers.
Immunohistochemical analysis showed enhanced nu-
clear staining for NF-nB/p65 in both high-grade
(P < .0001) and low-grade (P < .003) cancer speci-
mens, compared to benign tissue. The nuclear levels
of NF-nB/p65 correlated with concurrent increase in
cytosolic levels of InBa along with NF-nB–dependent
expression of Bcl2, cyclin D1, MMP-9, and VEGF. These
results demonstrate that NF-nB/p65 is constitutively
activated in human prostate adenocarcinoma and is
related to tumor progression due to transcriptional
regulation of NF-nB–responsive genes.
Neoplasia (2004) 6, 390–400
Keywords: Nuclear factor-nB, NF-nB – responsive genes, prostate cancer, InB, Rel A.
Introduction
Prostate cancer often progresses from an androgen-
dependent, organ-confined disease to a highly invasive,
androgen-independent malignancy with metastatic growth
properties [1,2]. Although localized forms of prostate cancer
can be effectively managed with surgery and other modalities,
no effective treatment is currently available for hormone-re-
fractory prostate cancer [3]. Considerable progress has been
made in the early detection and treatment of prostate cancer;
however, it has been difficult to distinguish between cancers
that will remain indolent and those that will behave aggres-
sively. Identification of predictive markers for prostate cancer,
especially those that are indicative of disease aggressiveness,
is important for improving the clinical management and sur-
vival of these patients.
Rel/nuclear factor-nB (NF-nB) is a dimeric transcription fac-
tor that plays important roles in the control of growth, differen-
tiation, and apoptosis [4,5]. It is also involved in immune and
adaptive responses to changes in cellular redox balance [6–8].
Rel/NF-nB consists of homodimers and heterodimers formed
by several subunits: NF-nB1 (p50/p105), NF-nB2 (p52/100),
Rel A (p65), Rel B, and c-Rel proteins [9]. These subunits have
a high level of sequence homology within the NH2-terminal
300 amino acids in the Rel homology domain [9,10]. The
inactive form of NF-nB is localized in the cytoplasm and con-
sists of three subunits: DNA-binding p50 and p65 subunits and
an inhibitory subunit, called InB, which is bound to p65 [10]. InB
masks the nuclear localization sequence and its release ini-
tiates activation of NF-nB and its subsequent translocation to
the nucleus, where it can bind to target sites in DNA [10].
Activation of NF-nB results in the induction of a large number of
genes that influence cellular proliferation, inflammation, and
cellular adhesion [11]. A number of critical genes that are
Abbreviations: NF-nB, nuclear factor-nB; NIK, NF-nB – inducing kinase; EMSA, electro-
phoretic mobility shift assay; MMP-9, matrix metalloproteinase-9; u-PA, urokinase-like
plasminogen activator; AP-1, activator protein-1; IL-6, interleukin-6; VEGF, vascular
endothelial growth factor; PSA, prostate-specific antigen; IAP, inhibitor of apoptosis;
COX-2, cyclooxygenase-2; NOS-2, nitric oxide synthase-2
Address all correspondence to: Sanjay Gupta PhD, Department of Urology, The James and
Eilleen Dicke Research Laboratory, Case Western Reserve University and University
Hospitals of Cleveland, Cleveland, OH 44106. E-mail: [email protected] work was supported by grants CA 94248 and CA 99049 from the United States Public
Health Service and funds from the Cancer Research and Prevention Foundation.2This work was conducted in The James and Eilleen Dicke Research Laboratory, Department
of Urology, Case Western Reserve University, Cleveland, OH.
Received 7 January 2004; Revised 4 February 2004; Accepted 4 February 2004.
Copyright D 2004 Neoplasia Press, Inc. All rights reserved 1522-8002/04/$25.00
DOI 10.1593/neo.04112
Neoplasia . Vol. 6, No. 4, July/August 2004, pp. 390 –400 390
www.neoplasia.com
RESEARCH ARTICLE
regulated by NF-nB include: anti–apoptotic genes (cIAP,
survivin, Bcl2, and BClxL), cell cycle– regulatory genes
(cyclin D1), genes encoding adhesion molecules, chemo-
kines, inflammatory cytokines, and genes involved in tumor
metastases (matrix metalloproteinase-9 [MMP-9], cyclooxy-
genase-2 [COX-2], nitric oxide synthase-2 [NOS-2], and
vascular endothelial growth factor [VEGF]) [4–7,11]. Inap-
propriate regulation of NF-nB and its dependent genes has
been associated with various pathologic conditions including
toxic/septic shock, graft-versus-host disease, acute inflam-
matory conditions, acute-phase response, viral replication,
radiation damage, atherosclerosis, and cancer [4–8]. Aber-
rant NF-nB activation has been implicated in the pathogen-
esis of several human malignancies including cancer of the
breast [12], colon [13], esophagus [14], gastrointestines [15],
liver [16], lungs [17], pancreas [18], skin [19], and uterine
cervix [20]. Studies have reported that NF-nB is constitutively
activated in human prostate cancer and prostate cancer
xenografts [21–24]. This activation occurs through signal
transduction pathways involving tyrosine kinases, NF-nB–
inducing kinase (NIK), and IKK, which ultimately leads to
phosphorylation and faster turnover of InBa, the superre-
pressor of NF-nB activation [21,22]. NF-nB has been shown
to activate a transcription-regulatory element of the prostate-
specific antigen (PSA)–encoding gene, a marker of prostate
cancer development and progression [25]. Increased NF-nB
activity in androgen-insensitive human prostate carcinoma
PC-3 cells contributes directly to its aggressive behavior [26].
Conversely, blockade of NF-nB activity in human prostate
carcinoma cells is associated with suppression of angiogen-
esis, invasion, and metastasis [27]. These findings suggest
that incessant activation of NF-nB in androgen-insensitive
human prostate carcinoma cells may contribute to aggres-
sive behavior; however, a definitive correlation between
NF-nB activation and tumor aggressiveness is lacking. We
undertook the present study to determine whether increased
NF-nB activation and its binding to DNA enhance tumor
aggressiveness and could predict the clinical outcome of
the disease. Using several techniques including immunohis-
tochemistry, Western blot analysis, and electrophoretic mo-
bility shift assay (EMSA), we found evidence of constitutive
activation of NF-nB/p65, its nuclear localization, increased
DNA binding, along with upregulation of NF-nB–regulated
gene increases with tumor grade. These results further
suggest that the NF-nB signaling pathway is a potential
target for therapeutic intervention.
Materials and Methods
Tissue Samples
Discarded benign and malignant prostate tissues from
patients undergoing surgery were obtained from the Tissue
Procurement Facility of Case Research Institute and the
Midwestern Division of the Cooperative Human Tissue Net-
work (Columbus, OH). The Gleason grade and score of
adenocarcinoma specimens were assigned by a surgical
pathologist experienced in genitourinary pathology. Immedi-
ately after procurement, samples were snap-frozen in liquid
nitrogen and stored at �80jC until further use. In addition,
4-mm tissue sections were obtained from 15 benign speci-
mens of the prostate and 49 other prostate cancer speci-
mens. Preoperative serum PSA levels were determined by
review of medical records. These studies were approved
by the Institutional Review Board at Case Western Reserve
University.
Western Blot Analysis
Frozen tissues (benign or cancer) were processed for
cytosolic and nuclear lysate, and the protein content was
determined using the DC Bio-Rad protein assay kit (Bio-Rad
Laboratories, Hercules, CA) as previously described [28].
For Western blot analysis, 25 mg of protein was resolved
using 4% to 20% polyacrylamide gels (Novex, Carlsbad, CA)
and transferred to a nitrocellulose membrane. The blot was
blocked in blocking buffer (5% nonfat dry milk/1% Tween-
20 in 20 mM TBS, pH 7.6) for 2 hours at room temperature;
incubated with mouse monoclonal antibodies of NF-nB/p65,
NF-nB/p50, InBa, Bcl2 (Santa Cruz Biotechnology, Santa
Cruz, CA), p-InBa (Cell Signaling Technology, Beverly, MA),
cyclin D1, MMP-9, and VEGF (Lab Vision Corp., Fremont,
CA) in blocking buffer for 2 hours at room temperature or
overnight at 4jC; followed by incubation with anti–mouse
IgG secondary antibody conjugated with horseradish perox-
idase (HRP; Amersham-Pharmacia, Piscataway, NJ) and
detected by ECL chemiluminescence and autoradiography
using XAR-5 film (Eastman Kodak, Rochester, NY). It is im-
portant to emphasize here that for detection of NF-nB/Rel
members, we used monoclonal antibodies that could detect
the activated form of NF-nB/Rel proteins by recognizing
an epitope overlapping the nuclear localization signal and
InBa-binding site of NF-nB/p65 and NF-nB/p50, and are
therefore selective and specific for the activated forms.
EMSA
EMSA for NF-nB was performed in the nuclear fraction of
prostate tissue using Lightshift Chemiluminiscent EMSA kit
(Pierce Biotechnology, Rockford, IL) following manufactur-
er’s protocol. Briefly, DNA was biotin-labeled using the biotin
3V end labeling kit (Pierce Biotechnology) in a 50-ml reaction
buffer, 5 pmol of double-stranded NF-nB oligonucleotide
(5V-AGTTGAGGGGACTTTCCCAGGC-3V and 3V-TCA
ACTCCCCTGAAAGGGTCCG-5V) incubated in a microfuge
tube with 10 ml of 5 � terminal deoxynucleotidyl transfer-
ase (TdT) buffer, 5 ml of 5 mM biotin-N4-CTP, 10 U of diluted
TdT, and 25 ml of ultrapure water at 37jC for 30 minutes.
The reaction was stopped with 2.5 ml of 0.2 M EDTA. To
extract labeled DNA, 50 ml of chloroform:isoamyl alcohol
(24:1) was added to each tube and centrifuged at 13,000g.
The top aqueous phase containing the labeled DNA was
further used for binding reactions. Each binding reaction
contained 1 � binding buffer (100 mM Tris, 500 mM KCl,
10 mM dithiothreitol, pH 7.5), 2.5% glycerol, 5 mM MgCl2,
50 ng/ml poly(dI–dC), 0.05% NP-40, 2.5 mg of nuclear
extract, and 20 to 50 fmol of biotin end-labeled target
DNA. The contents were incubated at room temperature
Rel A Is Activated in Human Prostate Adenocarcinoma Shukla et al. 391
Neoplasia . Vol. 6, No. 4, 2004
for 20 minutes. To this reaction mixture, 5 ml of 5 � loading
buffer was added, subjected to gel electrophoresis on a
native polyacrylamide gel, and transferred to a nylon mem-
brane. After transfer was completed, DNA was crosslinked
to the membrane at 120 mJ/cm2 using a UV crosslinker
equipped with 254-nm bulb. The biotin end-labeled DNA
was detected using streptavidin–HRP conjugate and a
chemiluminiscent substrate. The membrane was exposed
to X-ray film (XAR-5; Amersham Life Science, Inc., Arlington
Height, IL) and developed using a Kodak film processor
(Eastman Kodak). For supershift assay, antibodies against
NF-nB/p65 and NF-nB/p50 were added 30 minutes after
the beginning of the reaction, and incubation was continued
for an additional 30 to 45 minutes. DNA protein complexes
were analyzed as described earlier.
Immunohistochemistry
Immunohistochemical staining of paraffin-embedded sec-
tions was performed with a sensitive Dako EnVision+ System
(Dako Cytomation, Carpentaria, CA) according to the man-
ufacturer’s protocol. This is an extremely sensitive system
and offers an enhanced signal generation for the detection of
antigen. Briefly, 4-mm-thick paraffin-embedded sections from
benign and cancer tissues were deparaffinized, rehydrated,
immersed in target retrieval solution, and blocked for endog-
enous peroxidase activity. The sections were permeabilized
in TNB-BB (100 mM Tris, pH 7.5, 150 mM NaCl, 0.5%
blocking agent. 0.3% Triton-X, and 0.2% saponin) and
incubated in primary monoclonal antibodies of NF-nB/p65,
NF-nB/p50, and InBa (Santa Cruz Biotechnology) at 1:200
dilution, respectively, overnight at 4jC. Control sections
were incubated with antisera in the presence of 10-fold
excess of these antibodies or with isotype-matched IgG
normal goat serum. After washing three times in TBS,
sections were incubated for 2 hours at room temperature
with HRP-labeled polymer conjugated with secondary anti-
body. Immunoreactive complexes were detected using
AEC+ substrate chromagen consisting of 3-amino-9-ethyl-
carbazole. Slides were then counterstained in Mayer’s he-
matoxylin, mounted in crystal mount media, and dried
overnight on a level surface.
Evaluation of Immunostaining
The immunostained sections were examined indepen-
dently by three of the authors (S.S., S.G., and G.T.M.) using
light microscopy. Positive nuclear staining in high-grade
cancer cells was used as positive control for NF-nB staining.
Sections were examined with an inverted Olympus BH2 mi-
croscope (Olympus America Inc., Melville, NY) and images
were acquired with Image Proplus software (Media Cyber-
netics, Carlsbad, CA), digitally scored on a computer. The
intensity of staining was graded semiquantitatively and
each specimen was assigned a score on a scale from 0 to
3, designated as 0 (negative), 1 (weak), 2 (moderate), and
3 (strong). The immunoreactive score was designated by
the percentage of positive cells and the staining intensity as
previously described [29]. The percent of nuclear staining for
NF-nB/p65 was scored by counting the positive-stained cells
and the total number of cells quantified in random micro-
scopic fields (magnification, �400) with the assistance of
the software program.
Statistical Analysis
All measures were summarized as mean ± SD. Graphical
summaries of the distributions of measure were made using
boxplots. Kruskal-Wallis test, a nonparametric test based on
Wilcoxon scores, was used to examine the mean or median
difference of NF-nB/p65, NF-nB/p50, and InBa (using actual
values instead of levels) between two tissue groups. Differ-
ences in nuclear staining of NF-nB/p65 between benign
tissue and cancers of differing Gleason score (2 + 2, 3 + 3,
4 + 4, and 5 + 5) were also tested using Kruskal-Wallis test.
All tests were two-sided and P values less than .05 were
considered significant. All analyses were performed with the
SAS (Statistical Analysis System, version 6.12) software
(Cary, NC).
Results
To ascertain whether NF-nB is activated in human prostate
adenocarcinoma, we performed immunoblot analysis for
NF-nB/p65 and NF-nB/p50 in the nuclear fraction of benign
prostate tissue and prostate cancer specimens. As shown in
Figure 1A, immunoblot analysis for NF-nB/p65 in the nuclear
fraction exhibited significantly higher levels of protein ex-
pression in high-grade cancer tissue (Gleason score 5 + 4
and 5 + 5) compared with low-grade (Gleason score 3 + 3)
and benign tissue. However, no significant variation in the
protein expression of NF-nB/p50 was observed in these
tissues.
Next we analyzed the expression of NF-nB–inhibitory
protein, InBa, in the cytosolic fraction of benign prostate
tissue and prostate cancer specimens. As shown in
Figure 1A, immunoblot analysis for InBa exhibited a similar
pattern of protein expression as previously observed for
NF-nB/p65 with significantly high levels in high-grade cancer
tissue compared with low-grade cancer and benign tissue.
Because the rapid turnover of InBa requires phosphoryl-
ation at its N-terminal serine residues as a signal for ubiq-
uitination that eventually leads to its degradation, we
determined the phosphorylation of InBa in benign prostate
tissue and prostate cancer specimens. For this, we used an
antibody specific to phosphorylation sites at the N-terminal
Ser 32/36 of InBa. Compared to benign tissue, a progressive
increase in p-InBa with increasing tumor grade was ob-
served in prostate cancer specimens similar to the previously
noted increase in InBa (Figure 1A).
We next performed EMSA in the nuclear fractions
obtained from benign prostate tissue and prostate cancer
specimens. EMSA is a technique used to confirm the in-
creased nuclear translocation and DNA-binding activity of
NF-nB. As shown in Figure 1B, increased DNA-binding
activity was observed in prostate cancer tissue, compared
to benign tissue. The increase in DNA-binding activity was
more significant in high-grade cancer tissue compared with
392 Rel A Is Activated in Human Prostate Adenocarcinoma Shukla et al.
Neoplasia . Vol. 6, No. 4, 2004
low-grade cancer or benign tissue. To further confirm the
specificity of NF-nB DNA binding, we performed supershift
assay with antibodies specific for NF-nB/p65 and NF-nB/p50
and a competitive study with a 20-fold excess of unlabeled
oligonucleotide. As shown in Figure 1B, antibodies specific
for NF-nB/p65 (which recognizes Rel A homodimer) and
NF-nB/p50 supershifted complex I but were unable to shift
complex II, consisting of NF-nB/p50. Unlabeled oligonucle-
otide diminished the intensity of NF-nB/p65, indicating that
the shifted complex was a NF-nB–specific band (data not
shown) (Figure 1B).
We next examined the protein expression of NF-nB/p65,
NF-nB/p50, and InBa by immunohistochemical analysis in
benign prostate tissue and prostate cancer specimens.
Cancers were analyzed according to Gleason grade and
tissue specimens were assigned to three subgroups consist-
ing of benign tissue, low-grade cancer (Gleason score V 6),
and high-grade cancer (Gleason score 7–10). The staining
intensity was based on the scoring pattern of positive-stained
cells in the tissue specimens. Benign tissues did not exhibit
any significant NF-nB/p65 expression. In 15 specimens
analyzed, two exhibited moderate expression, six exhibited
weak expression, and seven were negative for NF-nB/p65
(Table 1). Most of the stainings observed in benign tissue
were nonspecific and localized in areas of inflammation
(Figure 3, A and B). A progressive increase in the staining
intensity of NF-nB/p65 with increasing tumor grade was
observed in the prostate cancer specimens. In 21 low-grade
tumors, 4 exhibited strong NF-nB/p65 expression, 10 ex-
hibited moderate staining, 5 exhibited weak staining, and
2 showed no NF-nB/p65 staining. In 28 high-grade tumors,
8 exhibited strong staining, 14 exhibited moderate staining, 3
exhibited weak staining, and 3 were negative for NF-nB/p65
staining (Table 1). The boxplots of NF-nB/p65 protein
exhibited a wide interindividual variation in cancer specimens
compared to benign tissue. The average scoring patterns
were as follows: benign tissue 0.72 ± 0.6 (mean ± SD); low-
grade tumor 1.66 ± 0.6 (mean ± SD); and high-grade tumor
Figure 1. (A) Protein expression of NF-jB/p65, NF-jB/p50, IjBa, and its phosphorylation in benign prostate tissue and prostate cancer specimens. Cytosolic and
nuclear extracts from tissues were prepared and electrophoresed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western
blotting with anti – p65 and anti – p50 antibodies in the nuclear fraction, and anti-IjBa and anti –p-IjBa in the cytosolic fraction. Cancer tissue is subdivided into low-
grade cancer (Gleason score V6) and high-grade cancer (Gleason score 7– 10). A progressive increase in the protein expression of NF-jB/p65 in the nuclear
fraction, and IjBa and p-IjBa in the cytosol, was observed in prostate cancer specimens. To ensure equal protein loading in the nuclear and cytosolic fractions, the
membrane was stripped and reprobed with anti – Oct-1 and anti –a-tubulin antibody. (B) NF-jB DNA-binding activity in benign and prostate cancer specimens by
EMSA. EMSA was performed to identify nuclear translocation of NF-jB dimers and their binding to DNA. An increase in the nuclear translocation of NF-jB/p65 was
observed in cancer tissue compared to benign tissue. NF-jB complexes I and II are indicated with arrows to the right of the panel. Supershift assay was performed
in high-grade tumor tissue with antibodies specific for NF-jB/p65 and NF-jB/p50. A shift in NF-jB/p65 was observed in high-grade tumor tissue. The details are
described in Materials and Methods section. Controls: (1) biotin –EBNA control DNA; (2) biotin –EBNA control DNA + EBNA extract; and (3) biotin –EBNA control
DNA + EBNA extract + 20-fold molar excess of unlabeled EBNA DNA.
Table 1. Expression of NF-nB/p65, NF-nB/p50, and InBa in Benign and Prostate Cancer Tissue Specimens.
NF-nB/p65
Specimen Type Number None Weak Moderate Strong P None Weak Moderate Strong P None Weak Moderate Strong P
Benign 15 7 6 2 0 13 2 0 0 8 4 3 0
Low-grade (V6) 21 2 5 10 4 18 3 0 0 4 7 6 4
High-grade (7 – 10) 28 3 3 14 8 .003* 16 6 4 2 .185 2 3 16 6 .002*
The expression of NF-nB/p65, NF-nB/p50, and InBa was evaluated on staining intensity of the tissues as: none (0), weak (1), moderate (2), and strong (3).
Fischer exact test was used to test the association between staining intensity and tissue type, or staining intensity and tumor grade.
*P value less than .05 was considered as significant.
NF-nB/p50 InBa
Rel A Is Activated in Human Prostate Adenocarcinoma Shukla et al. 393
Neoplasia . Vol. 6, No. 4, 2004
1.96 ± 0.8 (mean ± SD), respectively (Figure 2A). A signif-
icant increase in NF-nB/p65 score was observed in cancer
tissue compared with benign tissue, representing a 2.3-fold
increase in low-grade tumor (P < .003) and 2.7-fold increase
in high-grade tumor specimens (P < .0001) (Figure 2A).
Compared to benign tissue where negative to weak staining
was observed in most of the specimens, moderate to strong
staining was observed in the majority of cancers (Figure 3, A
and F ). Staining for NF-nB/p65 was observed both in the
cytoplasm and nucleus of tumor cells in low-grade tumor
(Gleason score 2 + 2), with a progressive increase in the
number of positive-stained cells with increasing tumor
grade (Gleason score 3 + 3) (Figure 3, C and D). Intense
and distinctly granular staining with increasing numbers of
positive stained nuclei was observed in the higher-grade
tumor specimens (Gleason score 4 + 4 and 5 + 5) (Figure 3,
E and F ).
We next examined the NF-nB/p50 protein expression in
benign prostate tissue and prostate cancer specimens.
Staining patterns for NF-nB/p50 in benign and cancerous
tissues were nonspecific; staining was limited to muscle
cells and scattered epithelial cells (Figure 4, A–D). In
15 benign specimens, 2 exhibited weak expression, where-
as 13 were negative for NF-nB/p50 expression. The staining
pattern for NF-nB/p50 in cancer specimens was predomi-
nantly weak to moderate and nonspecific. Weak expression
was noted in 3 of 21 specimens in low-grade tumor tumors
and 18 tumors were negative for NF-nB/p50 expression.
A similar staining pattern was observed in high-grade
tumors. Strong staining was observed in 2 of 28 specimens,
4 exhibited moderate staining, 6 exhibited weak staining,
and 16 were negative for NF-nB/p50 expression (Table 1).
The boxplots of NF-nB/p50 protein exhibited low scores,
implying that NF-nB/p50 does not participate in prostate
carcinogenesis. Average scoring patterns were as follows:
benign tissue 0.19 ± 0.2 (mean ± SD); low-grade tumor
0.22 ± 0.2 (mean ± SD); and high-grade tumor 0.66 ± 0.7
(mean ± SD), respectively (Figure 2B).
Next we examined the levels of cytosolic InBa protein
expression in the benign prostate tissue and prostate cancer
specimens. InBa is bound to the NF-nB/p65 in the cytoplasm
and inhibits its nuclear translocation. Benign tissue did not
exhibit any significant InBa expression. Moderate staining
was noted in 3 of 15 specimens, 4 exhibited weak staining,
and 8 were negative for InBa expression. Prostate can-
cer specimens showed significant staining for InBa, which
progressively increased as tumor grade increased. In 21 low-
grade tumor specimens, strong InBa expression was ob-
served in four specimens, six specimens exhibited moderate
staining, seven specimens exhibited weak expression, and
four were negative for InBa expression. Strong staining was
noted in 6 of 28 high-grade tumors, 16 exhibited moderate
staining, 3 exhibited weak staining, and 2 samples were neg-
ative for InBa expression (Table 1). The boxplots of InBa
protein displayed a wide interindividual variation in tumor
specimens compared to benign tissue. Average scoring
patterns were as follows: benign tissue 0.61 ± 0.6 (mean ±
SD); low-grade tumor 1.31 ± 0.8 (mean ± SD); and high-
grade tumor 1.88 ± 0.6 (mean ± SD), respectively (Figure
2C ). A significant increase in InBa score was observed in
tumor tissue compared with benign tissue, representing a
2.1-fold increase in low-grade tumor (P < .01) and 3.1-fold
increase in high-grade tumor specimens (P < .0001)
(Figure 2C). Compared to benign tissue (Figure 4E ), the
staining for InBa was predominantly observed in the cyto-
plasmic fraction in cancer specimens. Staining intensity
progressively increased with tumor grade (Figure 4, F–H ).
Next we extended our observation of increased constitu-
tive NF-nB/p65 activation in prostate cancer by quantification
Figure 2. Boxplots for (A) NF-jB/p65, (B) NF-jB/p50, and (C) IjBa based on
the staining pattern in benign prostate tissue and prostate cancer specimens.
The intensity of staining was graded semiquantitatively by assigning a score
to each tissue. Compared to benign tissue, staining was prominent for NF-jB/
p65 in low-grade (P < .003) and high-grade (P < .0001) cancer specimens.
Similar results were observed for InBa staining in low-grade (P < .01) and
high-grade (P < .0001) specimens, compared to benign tissue. No significant
difference in staining pattern between benign and cancer tissue was
observed for NF-nB/p50. NS, nonsignificant; ( I) Min – Max, ( ), 25% to
75%; (——) median value. The details are described in Materials and
Methods section.
394 Rel A Is Activated in Human Prostate Adenocarcinoma Shukla et al.
Neoplasia . Vol. 6, No. 4, 2004
of nuclear NF-nB/p65 expression in specimens of different
cancer grades. As shown in Figure 5, we counted the
number of stained cells and calculated the percentage of
positive-stained cells in benign and tumor specimens. Com-
pared to benign tissue, a significant increase in the percent-
age of cells with positive nuclear staining was observed with
increasing tumor grade that ranged from 13.5% in Gleason
score 2 + 2 to 17.6% in Gleason score 3 + 3, 25.2% in
Gleason score 4 + 4, and 44.8% in Gleason score 5 + 5,
respectively. Our findings suggest that NF-nB/p65 is activat-
ed in prostate cancer and its translocation to the nucleus
increases progressively in parallel with increasing tumor
grade.
Because NF-nB has been shown to regulate the expres-
sion of a number of genes whose products are involved in
carcinogenesis, we evaluated the protein expression of Bcl2,
cyclin D1, MMP-9, and VEGF, all of which are associated
with progression of prostate cancer. As shown in Figure 6, a
significant increase in the protein expression of Bcl2, cyclin
D1, MMP-9, and VEGF was observed in high-grade cancer
tissue compared with low-grade and benign tissue. Patterns
of increased expression of NF-nB–regulated proteins were
comparable to the observed increases in NF-nB/p65 and
InBa expression.
Discussion
One of the many challenges in the effective management of
prostate cancer is the identification of molecular marker(s)
capable of predicting tumor aggressiveness and disease
progression. Compelling evidence suggests that constitutive
activation of NF-nB is a hallmark of a number of human
Figure 3. Immunostaining for NF-jB/p65 in representative samples of benign prostate tissue and prostate cancer specimens of various Gleason grades.
Benign tissues showed no significant NF-jB/p65 staining (A and B). A progressive increase of NF-jB/p65 protein was observed in cancer specimens.
Low-grade cancer exhibited weak to moderate NF-jB/p65 protein expression (C and D), whereas strong staining was observed in high-grade cancer
specimens with more granular nuclear staining (E and F) (magnification, �200). The details are described in Materials and Methods section.
Rel A Is Activated in Human Prostate Adenocarcinoma Shukla et al. 395
Neoplasia . Vol. 6, No. 4, 2004
malignancies [30,31]. Aberrant NF-nB activation has been
implicated in the pathogenesis of many human diseases
including toxic/septic shock, graft-versus-host disease,
acute inflammatory conditions, viral replication, radiation
damage, and atherosclerosis [32,33]. Constitutive NF-nB
activation has been detected in several types of carcinoma;
Figure 4. Immunostaining for NF-jB/p50 and IjBa in representative samples of benign prostate tissue and prostate cancer specimens of various Gleason grade.
Benign and cancer specimens were negative for NF-jB/p50 staining (A –D). IjBa expression was weak to negative in benign tissue (E). A progressive increase in
IjBa protein was observed in cancer specimens. Low-grade cancer exhibited weak to moderate IjBa protein expression, (F) whereas strong staining pattern was
observed in high-grade cancer specimens, mostly in the cytoplasm of epithelial cells (G and H) (magnification, �200). The details are described in Materials and
Methods section.
396 Rel A Is Activated in Human Prostate Adenocarcinoma Shukla et al.
Neoplasia . Vol. 6, No. 4, 2004
mutations and rearrangements of NF-nB/InB family mem-
bers have been observed only in some hematologic cancers
[34,35]. In vivo activation of NF-nB/Rel members has been
definitively demonstrated in some forms of human malignan-
cies that include cancer of the breast [12], colon [13],
esophagus [14], gastrointestines [15], liver [16], lungs [17],
pancreas [18], skin [19], and uterine cervix [20], implicated
as part of the activated NF-nB complex. Studies conducted
on human prostate carcinoma cells have shown that NF-nB
is constitutively activated in androgen-insensitive DU145 and
PC3 cells, and prostate cancer xenografts [21–24]. A previ-
ous study has demonstrated NF-nB nuclear localization and
its prognostic significance in prostate cancer [23]; however, it
is not clear whether NF-nB activation reflects proliferative
activity of benign and malignant prostate epithelial cells, or
whether the activation that has been observed plays a role in
disease progression. We examined a large number of benign
and malignant human prostate tissues to evaluate the role of
NF-nB/InB activation in human prostate adenocarcinoma.
Our studies suggest that NF-nB/p65 (but not NF-nB/p50) is
constitutively activated in human prostate adenocarcinoma,
and that increasing levels of activation correlate with increas-
ing Gleason grade of cancer. We observed increased DNA-
binding activity of NF-nB/p65 in cancer tissue, as compared
with benign tissue, correlated with increased InBa expres-
sion in the cytoplasm. Previous studies have demonstrated
that Rel A/p65 exhibited strong transactivation potential as
observed by its constitutive activation in some forms of
human cancers [34]. Nuclear translocation of Rel A and
NF-nB–DNA-binding activity are higher in human tissues
from cervical cancer [20], colon adenocarcinoma [13], gastric
carcinoma [15], hepatocellular carcinoma [16], and pancre-
atic adenocarcinoma [18] compared to their normal counter-
parts. Similarly, nuclear translocation of Rel A–p50 complex
Figure 5. Score for nuclear staining of NF-jB/p65 in benign prostate tissue and prostate cancer specimens. Nuclear staining was determined by counting the
number of NF-jB/p65 – positive cells and total number of cells at �400 magnification. A progressive increase in the percentage of NF-jB/p65 cells was observed
as tumor grade increased. Compared to benign tissue, there is an increase in percent nuclear expression of NF-jB/p65 in low-grade cancer (Gleason score 2 + 2)
(P < .062), and a significantly higher level of percent nuclear expression (P < .0001) in high-grade cancer. The details are described in Materials and Methods
section.
Figure 6. Protein expression of NF-jB – regulated genes specifically Bcl2,
cyclin D1, MMP-9, and VEGF in benign prostate tissue and prostate cancer
specimens. Total cell lysates from tissues were prepared and electro-
phoresed by SDS-PAGE followed by Western blotting with anti –Bcl2, anti –
cyclin D1, anti –MMP-9, and anti – VEGF monoclonal antibodies. Cancer
tissue is subdivided into low-grade cancer (Gleason score V6) and high-
grade cancer (Gleason score 7 –10). A progressive increase in the protein
expression of Bcl2, cyclin D1, MMP-9, and VEGF was observed in prostate
cancer specimens. To ensure equal protein loading, the membrane was
stripped and reprobed with anti –a-tubulin antibody. The details are described
in Materials and Methods section.
Rel A Is Activated in Human Prostate Adenocarcinoma Shukla et al. 397
Neoplasia . Vol. 6, No. 4, 2004
occurs in human breast cancer tissues and derived cell lines
[12,36]; however, others have found that c-Rel, NF-nB1/p50,
NF-nB2/p52, and Bcl3, rather than Rel A, are the major
components in human breast cancer tissues [37]. Increased
Rel A activity and enhanced nuclear localization of p65–p50
dimer have been observed in melanoma and thyroid cancer
cells compared to normal equivalent cell lines [38,39]. Like-
wise, NF-nB/p50 has been noted to have low transactivation
activity and may have a limited role in carcinogenesis [40].
Constitutive activation of NF-nB/p50 has been observed in
non small cell lung carcinoma [17] and skin carcinogenesis
model [41]. In our studies, we found no significant NF-nB/p50
activation in prostate cancer tissue specimens.
Altered expression of InBa in cancer tissue has been
linked to constitutive NF-nB activation through phosphoryla-
tion of InBa at Ser32/36, resulting in the release and nuclear
translocation of active NF-nB [9,10]. We observed a pro-
gressive increase in the protein expression of InBa and its
phosphorylation in cancer specimens compared with benign
tissue, and the level of protein expression of InBa increased
in parallel with cancer grade. This marked increase in InBa
protein expression and its phosphorylation in cancer tissue
may be the consequence of functional activation of NF-nB in
prostate cancer, which is known to cause strong transcrip-
tional upregulation of InBa as a feedback mechanism possi-
bly operative in other cancers as well. Our results are in
agreement with previous observations that InBa protein
levels were increased in androgen-insensitive human pros-
tate carcinoma cells due to phosphorylation and faster
turnover of InBa in the cytosolic fraction [21,22]. It has been
shown that the upstream events associated with the consti-
tutive activation of NF-nB in prostate carcinoma cells involve
activation of tyrosine kinases NIK and IKK [21,22]. Studies
have further demonstrated that the tumor-suppressor PTEN
inhibits NF-nB activation and has been implicated in prostate
cancer [42]. The role of NF-nB in prostate cancer cells and
tissues has been previously reported, but no correlation has
been established between nuclear localization of NF-nB and
disease progression. In the present study, we observed that
nuclear staining of NF-nB/p65 is more pronounced in can-
cers of higher Gleason grade.
Accumulating evidence suggests that NF-nB has an
important role in carcinogenesis and tumor progression
[9,10]. NF-nB may be activated by cytokines, epidermal
growth factor, receptor expression, and Ras activation, or
through oxidative stress and cell damage [11]. Importantly,
chronic inflammation and release of proinflammatory cyto-
kines, known to induce NF-nB, could provide selective
growth advantage of neoplastic cells [6,7]. Furthermore,
NF-nB is involved in the regulation of several gene products
that may be important for cell motility and metastasis [11].
The expression of both MMP-9 and urokinase-like plasmin-
ogen activator (u-PA) is upregulated by NF-nB [42,43]. The
expression of cytokines IL-8 and IL-6, which are important for
cell migration, is also regulated by NF-nB [43]. Studies have
shown that the constitutive activation of NF-nB/p50-Rel A
and family members of activator protein-1 (AP-1) family, Fra-
1 and JunD, are essential for deregulated expression of IL-6
in prostate cancer [44]. Constitutive NF-nB activation in
human prostate carcinoma PC-3 cells has been shown to
be associated with invasive behavior [26]. Further, blockade
of NF-nB activity in these cell lines by transfection with
mutated InBa leads to inhibition of proangiogenic markers:
(VEGF, IL-8, and MMP-9) tumor invasion and metastasis
[27]. Our study, demonstrating positive correlation between
increasing Gleason grade, NF-nB/p65 protein expression,
and DNA binding/nuclear colocalization of NF-nB/p65, sug-
gests that NF-nB may be a useful predictive marker for
prostate cancer aggressiveness. This may be further corre-
lated with increase in protein expression of MMP-9 and
VEGF in cancer specimens observed in our studies.
Prostate cancer progression is often characterized by
aggressive locally invasive growth, metastasis, and, eventu-
ally, androgen independence [1,2]. The serum level of PSA
has been widely recognized as a clinical marker for prostate
cancer progression and has proven useful in monitoring
effectiveness of treatment [45]. In patients managed by
hormone ablation therapy, rising serum PSA levels typically
signify prostate cancer progression [45]. Recently, NF-nB
has been shown to bind a nB response element in the
promoter of the PSA gene that may be responsible for
increased serum PSA levels during prostate cancer progres-
sion [25]. However, in the present study, we did not observe
any significant correlation with serum PSA levels and NF-nB
expression (data not shown). Additional studies with a large
sample size of various tumor grades are required to validate
these findings.
NF-nB is an important regulator of cell proliferation
through its direct role in cell cycle progression [46]. Studies
have shown that NF-nB can stimulate transcription of cyclin
D1, a key regulator of G1 checkpoint control, mediated by
direct binding of NF-nB to multiple sites in the cyclin D1
promoter [47,48]. Amplification and/or overexpression of
cyclin D1 has been shown in several types of human cancers
[49]. Further studies have shown prognostic significance of
cyclin D1 in prostate adenocarcinoma and in the develop-
ment of androgen-independent disease [49,50]. Our studies
have shown a positive correlation between cyclin D1 expres-
sion during constitutive NF-nB activation in prostate cancer
specimens and suggest as an additional predictive marker
for prostate cancer progression.
Constitutive NF-nB activation is a mechanism of protec-
tion against diverse apoptotic stimuli, including chemother-
apy and radiation therapy [51,52]. Numerous anti–apoptotic
genes are regulated by NF-nB, including genes encoding
Bcl2-like proteins and inhibitors of apoptosis proteins [4,8].
Activation of NF-nB and its nuclear translocation leads to
the transcription of these anti–apoptotic genes [4,8]. Stud-
ies have shown that transcriptional regulation of Bcl2 by
NF-nB protects human prostate carcinoma cells from many
different apoptotic stimuli such as hormone ablation, radio-
therapy, chemotherapy, and immunotherapy [53]. Studies
have further demonstrated that blockade of InBa phoshor-
ylation inhibits NF-nB translocation and activation, and IL-6
production, and induces TNF-a–mediated apoptosis in both
androgen-dependent LNCaP and androgen-independent
398 Rel A Is Activated in Human Prostate Adenocarcinoma Shukla et al.
Neoplasia . Vol. 6, No. 4, 2004
PC-3 cells [27]. Our studies suggest that increase in Bcl2
expression correlates with DNA binding and nuclear local-
ization of NF-nB/p65 in prostate cancer specimens, and, as
such, suggest that Bcl2 expression along with NF-nB/p65
and InBa levels in prostate cancer may be useful as
predictive markers of disease progression. Studies are
required to identify mechanisms of NF-nB activation and
to further evaluate the distinct roles of NF-nB/InB family
members, to assess their usefulness in devising strategies
for the effective management of prostate cancer.
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