Nuclear Factor-κB/p65 (Rel A) Is Constitutively Activated in Human Prostate Adenocarcinoma and...

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.


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



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.



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.



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.



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.



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



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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