Download Expression ofNF-kBin ProstateCancer Lymph NodeMetastases

The Prostate 58:308 ^313 (2004)
Expression of NF-kB in Prostate Cancer Lymph
Node Metastases
Hazem Ismail A.,1 Laurent Lessard,2 Anne-Marie Mes-Masson,2
and Fred Saad1,2*
1
Urologic Oncology Research Group, Department of Urology,University of Montreal (CHUM),
Montreal,Quebec,Canada
2
CHUM Research Centerand Montreal Cancer Institute, Montreal,Quebec,Canada
INTRODUCTION. Nuclear factor-kB (NF-kB) is a transcription factor that transactivates
genes involved in the regulation of cell growth, apoptosis, angiogenesis, and metastasis. Our
aim was to assess NF-kB expression in lymph node (LN) metastases of prostate cancer.
METHODS. Immunohistochemical staining was performed using the p65 anti-NF-kB
antibody. Seventy-seven paraffin-embedded LN specimens obtained from 54 prostate cancer
patients were analyzed. Of the 54 patients, 32 had positive LN metastases, while 22 showed no
evidence of metastasis and were considered as controls. The overall percentage of NF-kBnuclear localization was assessed, as well as the intensity of staining.
RESULTS. Nuclear localization of NF-kB was significantly greater in the metastatic LN
group compared to controls. In patients with positive-LN metastases, 84.4% showed >10%
nuclear staining in tumor cells. Moreover, 64.4% of the malignant LN specimens had >10%
nuclear staining in lymphocytes compared to 0% in controls. Intensity of cytoplasmic and
nuclear staining was higher in the metastatic LN group than in controls (P < 0.01).
CONCLUSIONS. Nuclear localization/activation of NF-kB is up-regulated in prostate cancer
LN metastasis. Such up-regulation of NF-kB activity is observed in the tumor cells as well as in
the surrounding lymphocytes. Prostate 58: 308–313, 2004. # 2003 Wiley-Liss, Inc.
KEY WORDS:
NF-kB; lymph nodes; prostate; human
INTRODUCTION
The majority of prostate cancer deaths are due to
metastases that are resistant to therapy [1]. The exact
molecular events associated with carcinogenesis, progression, and metastasis in prostate cancer are still
unclear. However, it is well documented that pathogenesis of cancer metastasis comprise a series of
sequential and selective steps that include tumor cell
proliferation, angiogenesis, invasion, survival, and
growth in distant organs [2].
Nuclear factor-kB (NF-kB) is an inducible dimeric
transcription factor that belongs to the Rel/NF-kB
family [3,4]. Classical NF-kB transcription factor is
formed by the p50 and RelA (p65) proteins [5–8].
NF-kB activation involves its release from its inhibitor,
IkB, and its subsequent translocation from the cytoplasm to the nucleus, where it binds to cognate
sequences in the promoter region of multiple genes
2003 Wiley-Liss, Inc.
[3–8]. Regulation of gene expression by NF-kB is
controlled mainly by the inhibitory IkB proteins, which
include IkBa [8]. Upon stimulation, IkBa is rapidly
phosphorylated and degraded via the ubiquitin-proteasome pathway, permitting activation and nuclear
importing of NF-kB [8]. Numerous in vivo and in vitro
Grant sponsor: Prostate Cancer Research Foundation of Canada.
A.-M. M.-M. is a recipient of a Chercheur national fellowship from
the FRSQ L.L. is funded by a CIHR studentship.
*Correspondence to: Dr. Fred Saad, MD, FRCSC, Associate Professor
of Urology, Head of Urologic Oncology, Department of Urology,
Notre Dame Hospital, University of Montreal Health Center
(CHUM), 1560 Sherbrooke St. Montreal, Que., Canada H2L 4M1.
E-mail: [email protected]
Received 25 February 2003; Accepted 12 June 2003
DOI 10.1002/pros.10335
Nuclear Factor-kB (NF-kB) and Prostate Cancer
studies have shown that NF-kB/RelA plays an important role in the regulation of cell proliferation,
apoptosis, adhesion, and growth [9–11]. Furthermore,
NF-kB has been shown to regulate the expression of
various angiogenic factors including vascular endothelial growth factor (VEGF), interleukin (IL)-8, and type
IV collagenases (MMP-2 and MMP-9) [13,14]. These
factors are essential for angiogenesis, as well as tumor
cell motility, invasiveness, and/or metastasis [13–15].
It has been recently reported that inhibition of NF-kB
activity in PC-3 cells was associated with decreased
expression of VEGF, IL-8, and MMP-9 [16]. Interestingly, inhibition NF-kB activity resulted in decreased
angiogenesis, invasion, and metastasis of human prostate cancer cells grown the prostate of nude mice [17].
We previously reported that nuclear localization of
NF-kB in prostate cancer tissues can be used in
correlation with Gleason score as predictors for poor
outcome, and that NF-kB activation correlated with
bone metastases, and hence poor prognosis [18].
These data suggest that NF-kB expression might
have an important role in the development of metastatic prostate disease. However, to the best of our
knowledge, local NF-kB expression in prostate cancer
lymph node (LN) metastasis has not been well documented. Our objective was to identify NF-kB expression in LN of patients with metastatic prostate disease.
MATERIALS AND METHODS
Tissues
LN specimens were obtained from 54 patients with
localized prostate cancer, who had undergone radical
prostatectomy. No patients received hormonal treatment prior to surgery. Of the 54 patients, 32 had
evidence of prostate cancer LN metastasis as confirmed
by PSA staining, while 22 had no evidence of metastasis
and were considered as controls. From the 32 metastatic
prostate cancer patients, 55 LN specimens were evaluated, of which 45 were positive for metastasis, while
ten were negative and were evaluated as internal
controls. In addition, 32 LN specimens from the
22 patients with no evidence of metastasis were
studied. One to two LN specimens were evaluated for
each of the 54 patients.
Immunohistochemistry
Tissue sections were immunostained for NF-kB using
the biotin–streptavidin immunoperoxidase method as
previously described [18]. Briefly, paraffin-embedded
LN sections (4 mm) were initially deparafinized with
toluene and rehydrated through graded ethanol. All
steps were performed at room temperature. Following
each step, sections were washed with 0.01 M phosphate
309
buffered saline (PBS solution) for 10 min. Endogenous
peroxidase activity was blocked with 3% hydrogen
peroxide/50% methanol for 15 min. Tissue sections
were incubated with a protein blocking serum-free
reagent (Dako Diagnostics, Inc., Ont., Canada) for 15 min
to block non-specific binding. An antigen retrieval
technique was applied for NF-kB by boiling slides for
10 min at 958C in a 0.01 M sodium citrate buffer (pH 6.0).
NF-kB expression was studied using monoclonal NF-kB
p65 (F-6) antibody (Santa Cruz Biotech, Santa Cruz, CA)
that recognizes the amino-terminal sequences of the p65
subunit. The NF-kB primary antibody was applied at a
concentration of 1:50 in PBS and was incubated for
60 min at room temperature. Immune complexes were
revealed using a biotin-conjugated broad spectrum
secondary antibody (20 min), then streptavidin-peroxidase conjugate for 20 min (Dako Diagnostics, Inc.),
followed by chromogen (0.06% 3,30 -diaminobenzidine
tetrahydrochloride, 0.01% hydrogen peroxide in PBS).
Sections were counter stained with Mayer’s haematoxylin, dehydrated, and then mounted. Negative controls
were included by omitting the primary antibody, while
positive controls were included using prostate tissue
sections.
Quantif|cation
LN sections were evaluated using light microscopy.
NF-kB localization within the cell cytoplasm and/or
nucleus was identified. Nuclear localization of NF-kB
was categorized as positive or negative, as well as an
overall proportion of cells (<1%, 1–10%, and >10%)
with positive nuclear staining in the studied field at
100 magnification [18]. For the positive-metastatic LN
group, tumor cells and the surrounding lymphocytes
were evaluated separately. A minimum of five different areas for each specimen were evaluated, and the
mean was assessed. The intensity of NF-kB staining
was evaluated semi-quantitatively on a scale ranging from 1 to 4þ. Statistical analysis was performed
using the w2-test.
RESULTS
The mean age of prostate cancer patients with
LN metastases was 64.2 4.7 years as compared to
65.1 4.3 years in the control group (P ¼ 0.6). The mean
Gleason score was similar in both groups (Gleason 8–
10; P ¼ 0.9), while the mean preoperative prostate
specific antigen (PSA) was 18.3 7.8 mg/L in the
metastatic prostate cancer patients compared to
17.6 7.1 mg/L in controls (P ¼ 0.8). NF-kB staining
was identified in the cytoplasm of lymphocytes in both
groups, as well as in the malignant epithelial cells of the
tumor-positive LN group. In contrast, nuclear localization of NF-kB was variably expressed (Fig. 1).
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Ismail et al.
(P ¼ 0.8), which was significantly different from the
positive-LN specimens from the same patients
(Table II).
The intensity of NF-kB staining in the LN specimens
of metastatic prostate cancer patients and controls is
shown in Table III. While 68.8% of patients with
positive-LN metastases had 3 to 4þ intensity of staining
in both lymphocytes and malignant cells, only 9.1% of
the control group showed strong staining in the
lymphocytes. It is noteworthy to mention that, in
>90% of the metastatic LN specimens up-regulation
of NF-kB activation and expression was observed
simultaneously in lymphocytes and malignant cells.
In all examined specimens, nuclear localization of NFkB was persistently observed in lymphocytes and/or
malignant cells in the vicinity of lymphatic or vascular
channels.
DISCUSSION
Fig. 1. Nuclear factor-kB (NF-kB) immunostaining patterns in
lymphnodes (LN) ofprostate cancerpatients.Patientswithpositive
LN metastases showed nuclear and cytoplasmic NF-kB staining in
(A) malignant acini (400), (B) lymphocytes (400), (C) malignant
acini and surrounding lymphocytes (400), and (D) cells in the
vicinity of lymphatic and vascular channels (staining of lymphocytes
atlymphatics and vascular channels was similar in controls; data not
shown).NF-kB-cytoplasmic staininginlymphocytes ofpatientswith
no metastasesis shownin (E) (200) and (F) (400).
Table I shows NF-kB-nuclear localization in patients
with positive LN metastases, where 27/32 (84.4%) of
patients had >10% nuclear staining in tumor tissues as
compared to 9.3 and 6.3% with <1% and 1–10% nuclear
staining, respectively (P ¼ 0.001; Fig. 1A,B). Lymphocytic-nuclear localization of NF-kB of >10% was seen in
68.7% of patients with positive-LN metastases and 0%
of controls (P ¼ 0.0001, Table II, Fig. 1C,D). The negative
LN specimens from patients with metastatic prostate
cancer (internal controls; n ¼ 10) showed similar
lymphocytic NF-kB-nuclear localization as controls
In the present study, we report for the first time NFkB expression in prostate cancer LN metastasis. Our
data shows that NF-kB nuclear localization/activation
is up-regulated in tumor cells, as well as in the
surrounding lymphocytes of metastatic LN of prostate
cancer patients. It is well documented that Gleason
score is an important prognostic marker for prostate
cancer metastasis [19,20]. In addition, NF-kB has been
reported to regulate the expression of PSA [21], another
important marker for prostate cancer progression.
Hence, the control group included in this study was
selected to match the positive-LN group with respect to
age, preoperative PSA, and Gleason score. Furthermore, to minimize the effect of known and/or
unknown coexisting variables that may be responsible
for increased NF-kB activity, LN with no evidence of
malignancy, as confirmed by PSA staining, from
patients with LN metastases were evaluated separately
as internal controls. The internal control group showed
similar NF-kB behavior as controls, which was significantly different from the positive-metastases LN
group. Based on these observations, we hypothesize
that the increased expression of NF-kB and its
subcellular localization in metastatic LN is directly
TABLE I. Nuclear Factor-kB (NF-kB)-Nuclear Localization in Tumor Cells of Patients
With Positive-Prostate Cancer Lymph Node (LN) Metastases
Percentage of NF-kB nuclear localization
Patients (n ¼ 32)
Specimensa (n ¼ 45)
a
<1%
1–10%
>10%
P value
3 (9.3%)
5 (11.1%)
2 (6.3%)
8 (17.8%)
27 (84.4%)
32 (71.1%)
0.001
0.001
One to two specimens were evaluated for each patient.
Nuclear Factor-kB (NF-kB) and Prostate Cancer
311
TABLE II. NF-kB-Nuclear Localization in the Lymphocytes of Patients With Prostate Cancer LNMetastases and Controls
Metastatic patients
NF-kB nuclear
staining
<%
1–10%
>10%
P valuea
Patients
(n ¼ 32)
Positive-LN
specimens (a)
(n ¼ 45)
Negative-LN
specimens (b)
(n ¼ 10)
3 (9.4%)
7 (21.9%)
22 (68.7%)
0.005
6 (13.3%)
10 (22.2%)
29 (64.4%)
0.003
7 (70%)
3 (30%)
0
0.001
Controls
P value
(a vs. b)
Patients
(n ¼ 22)
Specimens (c)
(n ¼ 32)
P value
(a vs. c)
0.001
0.5
0.001
17 (77.3%)
5 (22.7%)
0
0.001
24 (75%)
8 (25%)
0
0.001
0.001
0.9
0.0001
a
Significance between <1 and >10% NF-kB localization in patients and specimens.
related to the process of malignant cell progression and
ability to metastasize. This might in part explain our
observation of persistent nuclear-NF-kB expression in
malignant cells and lymphocytes in the vicinity of
lymphatic and vascular channels.
NF-kB has been implicated in the development,
activation, and function of B and T lymphocytes [22].
NF-kB importance for B and T cell function was proven
by the fact that lymphocytes lacking either one of the
two major inducible NF-kB transcription activators, CRel and RelA, have B and T cell proliferation defects
in response to certain stimuli [23–25]. Hence, we
observed diffuse cytoplasmic expression of NF-kB in
all lymphocytes of the metastatsis-positive LN group
and controls. However, significant up-regulation of
NF-kB nuclear expression was observed in the metastatic LN group especially in the lymphocytes surrounding malignant cells. It is noteworthy to mention
that none of our positive-LN specimens had histological features suggestive of hyperplasia and/or an
inflammatory reaction that might be responsible for
the lymphocytic activation of NF-kB.
Several studies have previously suggested that
NF-kB activation is associated with oncogenesis in
mammalian systems [26–36]. Amplification and
over expression of genes coding for RelA/NF-kB
factors have been found in leukemia [26], lymphoma
TABLE III. Intensity of NF-kB Staining in Patients With
Positive-Prostate Cancer LNMetastases and Controls
Intensity of
NF-kB staining
1þ
2þ
3–4þ
P valuea
a
Patients with LN
metastases (n ¼ 32)
Controls
(n ¼ 22)
3 (9.4%)
7 (21.9%)
22 (68.8%)
0.001
15 (68.2%)
5 (22.7%)
2 (9.1%)
0.002
P value
0.005
0.8
0.001
Significance between 1þ and 3–4þ intensity of NF-kB staining in
both lymphocytes and malignant cells of metastatic patients and
lymphocytes of controls.
[27], pancreatic adenocarcinoma [28], hepatocellular
carcinoma [29], thyroid C-cell tumor [30], and breast
cancer [31]. Furthermore, constitutive activation of
NF-kB has been reported to be a common characteristic
of many cell lines from hematopoietic and solid tumors
[5,28,29]. The inactivation of NF-kB in carcinoma cell
lines by different approaches has been shown to
dramatically reduce their ability to form colonies in
agar, as well as to grow in vivo and in vitro [32,33].
NF-kB has been also shown to have a key role in cell
protection against diverse apoptotic stimuli including
chemotherapeutic drugs and g-irradiation through
activation of the anti-apoptotic gene program in cells [5].
With respect to prostate cancer, NF-kB activation has
been detected in a large number of prostate cancer cell
lines, such as the androgen-resistant PC-3 and DU145
cell lines [12,34,35]. The tumor suppressor PTEN which
inhibit NF-kB activation, has been implicated in
prostate cancer [36]. In addition, it has recently been
shown that bcl-2 gene expression induced by tumor
necrotic factor (TNF)-a in LNCAP cells is mediated by
NFkB binding to specific sites in the bcl-2 P2 promoter
[37]. A possible role for NF-kB in regulating apoptosis
in androgen-independent prostate cancer cells has also
been suggested by the fact that inhibition of NF-kB
activity is necessary, although not sufficient, for the
induction of apoptosis [38]. The IL-6 gene, a target of
NF-kB, has been shown to be overexpressed in
localized prostate cancer and is thought to promote
prostate cancer cell growth [39]. Furthermore inhibition of NF-kB activity in PC-3 cells was reported to
be associated with decreased expression of various
potent angiogenic factors, such as VEGF, IL-8, and
MMP-9 [16]. Recently, it has been reported that
inhibition of NF-kB activation by dehydroxymethylepoxyquinomicin (DHMEQ) results in suppression of
hormone-refractory prostate cancer cells growth in
nude mice [40]. Moreover, it has been stated that
blockage of NF-kB activity is associated with reduced
angiogenesis, invasion, and metastasis of human
prostate cancer cells grown in vivo [17]. We previously
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Ismail et al.
reported that NF-kB-nuclear localization/activity in
primary prostate cancer tissues correlates with poor
patient outcome and bone metastasis [18]. The present
study is in agreement with all these observations, as we
were able to identify increased nuclear localization of
NF-kB in prostate cancer LN-metastases. These results
support and expand the possible role played by NF-kB,
and its activators, in the development of metastatic
prostate disease. However, further studies are essential
to confirm such a hypothesis, as well as to correlate
metastatic NF-kB expression to disease progression,
and its possible implication as a predictor of poor
prognosis. In addition, it would be interesting to determine whether our novel observation on NF-kB activation can correlate to clinical parameters such as time to
recurrence and survival. Moreover, studies aiming at
revealing the exact mechanisms by which the activation
of NF-kB contributes to metastasis are essential.
Finally, this study in conjunction with previously published reports [16–18] support the notion that targeting
and/or blocking NF-kB signaling in positive-malignant cells may be a valid therapeutic strategy in the
management of prostate cancer.
CONCLUSIONS
We report for the first time NF-kB expression in
prostate cancer LN metastases. Nuclear localization/
activation of NF-kB is up-regulated in metastatic LN.
Such observation suggests, and further supports, the
possible role played by NF-kB in the development of
metastatic prostate disease.
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