Independent role of phosphoinositol-3-kinase (PI3K) and Casein Kinase II (CK-2) in EGFR and Her-2-mediated constitutive NF-kappaB activation in prostate cancer cells

Abstract

Recent research has highlighted the potential role of EGFR and Her-2 in the constitutive activation of NF-kappaB (NF-kappaB) in prostate cancer cells, although the mechanism by which these receptors activate NF-kappaB in these cells remains unclear.
Using pharmacological and genetic approaches we show that in PC-3 cells, EGFR and Her-2 are involved in the constitutive activation of NF-kappaB through two different mechanisms. EGFR activates NF-kappaB through the PI3K/Akt pathway that leads to the phosphorylation of IkappaBalpha on serines 32 and 36, thereby promoting the nuclear translocation of the p65 subunit. In contrast, Her-2 activates NF-kappaB through Casein Kinase II (CK-2) activation independently of IkappaBalpha phosphorylation on serines 32 and 36.
Our study not only directly clarifies the signaling pathways involved in NF-kappaB activation in prostate cancer cell lines and but also provides a framework for further studies in the clinical characterization and management of prostate cancer.

Full text

The Prostate 65:306 ^315 (2005)
Independent Role of PhosphoInositol-3-Kinase (PI3K)
and Casein Kinase II (CK-2) in EGFR and
Her-2-Mediated Constitutive NF-kappaB
Activation in Prostate Cancer Cells
Ce
´cile Le Page,
1
Ismael Herve
´Koumakpayi,
1
Laurent Lessard,
1
Fred Saad,
1,2
and Anne-Marie Mes-Masson
1,3
*
1
Centre de Recherche du Centre Hospitalier del’Universite
¤de Montre
¤al (CR- CHUM) and Institut du cancer de Montre
¤al,
1560 rue Sherbrooke est, Montre
¤al, Que
¤bec,Canada
2
De
¤par teme nt d’urologie, Universite
¤de Montre
¤al, Montre
¤al, Que
¤bec,Canada
3
De
¤partement de Me
¤decine,Universite
¤de Montre
¤al, Montre
¤al, Que
¤bec,Canada
BACKGROUND. Recent research has highlighted the potential role of EGFR and Her-2 in the
constitutive activation of NF-kappaB (NF-kB) in prostate cancer cells, although the mechanism
by which these receptors activate NF-kB in these cells remains unclear.
METHODS AND RESULTS. Using pharmacological and genetic approaches we show that in
PC-3 cells, EGFR and Her-2 are involved in the constitutive activation of NF-kB through two
different mechanisms. EGFR activates NF-kB through the PI3K/Akt pathway that leads to the
phosphorylation of IkBaon serines 32 and 36, thereby promoting the nuclear translocation of the
p65 subunit. In contrast, Her-2 activates NF-kB through Casein Kinase II (CK-2) activation
independently of IkBaphosphorylation on serines 32 and 36.
CONCLUSIONS. Our study not only directly clarifies the signaling pathways involved in NF-
kB activation in prostate cancer cell lines and but also provides a framework for further studies
in the clinical characterization and management of prostate cancer. Prostate 65: 306 –315, 2005.
#2005 Wiley-Liss, Inc.
KEY WORDS: ErbB-1; ErbB-2/c-neu; signaling; PC-3; LNCaP
INT RODUCTION
In men, prostate cancer is the most frequently
diagnosed cancer and a leading cause of cancer death.
Treatment of prostate cancer depends on its stage. If the
tumor is localized and the patient is healthy, aggressive
therapy is often recommended. However, some early
stage tumors will remain latent and will not require
aggressive therapy, while others at risk of progression
need to be treated early. Unfortunately, there are no
known molecular markers that reliably predict if a
cancer will progress to an aggressive disease. It is
unclear which genes are implicated in the initiation and
progression of prostate cancer and to date attention has
focused on several genes and pathways involved in this
mechanism although few of these players are fre-
quently altered in primary prostate cancer. Recently, an
alternate candidate marker, Nuclear Factor-kappaB
(NF-kB) has been shown to play a role in the devel-
opment of prostate cancer and the progression to an
advanced disease [1–3].
Grant sponsor: Canadian Prostate Cancer Research Foundation;
Grant sponsor: The Canadian Uro-Oncology/Astra Zeneca Award;
Grant sponsor: Chercheur National fellowship from the Fonds de la
Recherche en Sante
´du Que
´bec (to A.-M.M.-M.); Grant sponsor:
Canadian Institute of Health Research/Canadian Prostate Cancer
Research Initiative studentship (to L.L.); Grant sponsor: The
Canderel fund of the Institut du Cancer de Montre
´al (I.H.K.).
*Correspondence to: Dr. Anne-Marie Mes-Masson, Centre de
recherche CHUM/Institut du cancer de Montre
´al, 1560 Sherbrooke
Est, Montre
´al, Que
´bec, Canada, H2L 4M1.
E-mail: anne-marie.mes-masson@umontreal.ca
Received 9 February 2005; Accepted 7 April 2005
DOI 10.1002/pros.20291
Published online 13 July 2005 in Wiley InterScience
(www.interscience.wiley.com).
2005Wiley-Liss,Inc.

The Rel/NF-kB transcription factor family is com-
posed of structurally related members that possess a
N-terminal Rel-Homology (RH) domain involved in
protein–protein interactions and DNA-binding. NF-kB
proteins exist as homo- and heterodimers expressed
in many cells but kept inactive and maintained in
the cytoplasm by IkB family of inhibitors. Classically,
the activation of NF-kB requires signals that converge
to the trimeric IkB kinase complex (IKK) resulting in
phosphorylation of IkBaon serine 32/36 and its subse-
quent ubiquitinylation and degradation allowing NF-
kB to translocate to the nucleus. The precise mechanism
by which NF-kB is constitutively activated in hormone-
independent prostate cancer cells is not well known.
Constitutive activation of PI3K/Akt pathway and IKK
has been implicated in this activation in the PC-3
hormone-independent prostate cancer cell line [1,4 –6].
PI3K activation results in the translocation of Akt from
the cytoplasm to the inner membrane where Akt is
phosphorylated on serine 473 and threonine 308 by
upstream kinases, PDK and ILK [7]. The activation of
Akt leads to the phosphorylation of downstream target
such as IKK, GSK3, Bad, caspase 9, and FOXO family of
forkhead transcription factors. However, it has yet to be
determined which initial event constitutively activate
PI3K/Akt pathway in prostate cancer cells.
In different epithelial cancer cell lines, evidence
points to a role of EGFR and Her-2 as upstream acti-
vators of the PI3K/Akt pathway and responsible for the
constitutive activation of NF-kB through the activation
of IKK [8–10]. EGFR and Her-2 are members of the
ErbB growth factor receptor family composed of four
distinct receptors: EGFR/ErbB1, Her-2/ErbB2/c-neu,
Her-3/ErbB3, and Her-4/ErbB4. In PC-3 cells, EGFR
and Her-2 overexpression and constitutive activation
modulate NF-kB signaling through two distinct mecha-
nisms: one dependent and one independent of the
phosphorylation of IkBaon serines 32/36 [11]. How-
ever the signaling intermediates that lead to this
constitutive NF-kB activation are presently not known.
Here, we investigated the role of PI3K/Akt pathway as
a downstream signaling component of EGFR and Her-2
in PC-3 cells. We observed that EGFR, but not Her-2,
activated NF-kB through the PI3K/Akt pathway,
which induces the phosphorylation of IkBaon serines
32/36 and thereby the nuclear translocation of NF-kB.
Then we further investigated the role of CK-2 in Her-2-
mediated signaling. CK-2 is an ubiquitous serine/
threonine kinase composed of two catalytic subunits
(aand a0) and one regulatory subunit (b). The regu-
lation and function of CK-2 is not well understood.
Originally CK-2 was defined as a constitutively active
protein kinase though recently several reports sug-
gest that CK-2 acts as a regulated kinase participa-
ting in signal transduction [12] and activation of NF-kB
[13–16]. Here we demonstrate for the first time a role
for CK-2 in Her-2–mediated NF-kB signaling.
MATERIALS AND METHODS
Cell Culture and Reagent
PC-3 and LNCaP cells were grown at 378C and in 5%
CO
2
in RPMI supplemented with 10% fetal bovine
serum (FBS) and 2 mg/ml gentamicin. Experiments
were performed on cells before passage 20. All reagents
used for cell culture media were from Gibco BRL. All
pharmacological inhibitors were from Calbiochem and
resuspended in dimethyl sulfoxide (DMSO).
Western Blot Analysis
Cells were lyzed with cold lysis buffer (10 mM Tris-
HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM DTT/
1 mM NaF/0.5% NP-40/0.5 mM PMSF/0.2 mM
sodium orthovanadate/2 mg/ml of aprotinin, leupep-
tin, and pepstatin), boiled in loading buffer, separated
by SDS–PAGE, and transferred on a nitrocellulose
membrane under refrigerated conditions (200 mA, 2 h).
Membranes were saturated with 5% milk/ PBS/0.1%
Tween 20. Immunodetection was done as described in
the protocol of the ECL kit (Amersham Biosciences UK
limited, England): i.e., incubated 2 hr at room tempera-
ture with the specific antibody (0.5 to 1 mg/ml), washed
two times with PBS/0.05% Tween 20 and incubated for
another 30 min at room temperature with peroxidase
conjugated antibodies (Santa-Cruz, CA). For Western-
blot polyclonal antibodies IkBa(B-8), c-myc (A-14), CK-
2a(C-18), CK-2a0(C-20), Akt-1 (D-17) from Santa Cruz
Biotechnology (CA), and phospho-IkBaand phospho-
Akt (Cell Signaling, MA), were used. Monoclonal
antibodies NF-kB p65 (F-6) and Ran (C-20) from Santa
Cruz, and beta-actin (ab6272, Abcam, MA) were used.
All experiments were repeated at least two times with
similar results.
Cytoplasmic and Nuclear Extracts
To prepare cellular extracts, 5 10
6
cells were wash-
ed twice in cold PBS and resuspended in 300 ml of lysis
buffer containing 10 mM Tris pH 7.9/10 mM NaCl/
5mMMgCl
2
/10 mM sodium orthovanadate/0.5 mM
PMSF/10 mg/ml of the protease inhibitors (PMSF,
pepstatin, leupeptin, and aprotinin). After swelling
cells for 30 min on ice, 0.1% Nonidet P-40, and 10%
glycerol (v/v) were added and lysed cells were centri-
fuged for 1 min at 48C and 5,000 rpm. Supernatants
were carefully decanted for cytoplasmic extracts.
Nuclei pellets were resuspended for 1 hr in 40 mlof
lysis buffer containing 10 mM Tris pH 7.9/400 mM
NaCl/0.1 EDTA/0.5 mM DTT/5% glycerol/0.5 mM
Mechanism of NF-kB Activation in Prostate Cancer Cells 307

PMSF/10 mg/ml protease inhibitors. Particulate matter
was eliminated by centrifugation for 10 min at
13,000 rpm at 48C. Protein concentration was deter-
mined using the Bradford method.
Electrophoretic Mobility Shift Assay (EMSA)
Nuclear cell extracts were prepared using 5 
10
6
cells incubated with the indicated inhibitors or
with vehicle alone. Ten micrograms were subjected
to EMSA using a double-stranded (50-AGTTGAG-
GGGAGATTTGCAGGC-30)
32
P-labelled probe that
corresponds to the NF-kB-binding element from the
Igk-chain promoter. Binding reactions were carried out
on ice for 30 min in binding buffer (20 mM Tris pH 8.0,
5% glycerol, 0.1 KCl, 0.2 mM EDTA pH 8.0, 0.2 EGTA
pH 8.0, 0.5 mg poly(dIdC). The resulting protein –DNA
complexes were separated on 5% non-denaturating
polyacrylamide gel in Tris-Glycine–EDTA buffer. Gels
were dried and exposed for autoradiography. All ex-
periments were repeated two times with similar
results.
Transfection and Luciferase Reporter Assay
Cells were co-transfected in 96-well plates with
0.2 mg of DNA and 200 ng of a constitutively active
Renilla luciferase (pCMV-RL, Promega, WI) by lipo-
fectamine method (Invitrogen, CA). After 6 hr, cells
were washed in fresh medium and incubated over-
night. Cells were then stimulated for 16 hr with the
different inhibitors or DMSO. Cells were then assayed
for luciferase activity using the dual luciferase reporter
assay system (Promega, WI). The 3enh-kb-CONA-luc,
carrying a firefly luciferase gene under the control of a
trimer of kB consensus was a gift from Dr. Juana
Wietzerbin (Paris, France). The PCDNA3-HERCD533
plasmid was a kind gift from Dr. Alex Ullrich
(Germany) and Dr. Sylvain Meloche (Montreal, Qc,
Canada). PCDNA3.1-Myc-mut-Her-2 was a generous
gift from Dr. Ming-Fong Lin (Omaha, Nebraska, USA).
CK-2a0was provided by Dr. David Lichfield (Ontario,
Canada). All experiments were repeated three to four
times with similar results.
CK-2 Kina se Assays
The CK-2 kinase assay was performed following the
instruction of the manufacturer (Upstate Biotechnol-
ogy Inc.). Briefly, 5 10
6
cells were washed and resus-
pended in lysis buffer containing 10 mM HEPES
pH 7.5/250 mM NaCl/5 mM MgCl
2
/20 mM sodium
orthovanadate/0.5 mM PMSF/ b-glycerophosphate/
1 mM NaF/2 mg/ml of the protease inhibitors
(pepstatin, leupeptin, and aprotinin). Samples (5 –
10 mg) were resuspended in kinase buffer and the assay
was performed at 378C for 10 min, with 200 mMof
specific peptide in the presence of PKA inhibitors and
0.5 mCi [
32
P]g-ATP. The resulting products were anal-
yzed on P81 filters, washed, and counted in scintillation
liquid. Counts in assay containing sample without
peptide were subtracted as background to calculate the
specific activity in each sample. All experiments were
repeated two to three times with similar results.
RESULTS
EGFR but not Her-2 Activates the PI3K/Akt
Pathway in PC-3 and LNCaP Cells
The PI3K/Akt pathway can be activated by various
growth factors, including EGF through the activation of
ErbB receptors [17]. To investigate the role of EGFR
and Her-2 on PI3K/Akt pathway activation in prostate
cancer cells, we tested the effect of ErbB inhibitors on
the Akt phosphorylation in the PC-3 and LNCaP cell
lines. The efficiency of these inhibitors had already
been tested and described in our different prostate
cancer cell lines [11] and confirmed the results reported
by others [18,19]. Under appropriate conditions,
tyrphostins AG1517 and AG1478 selectively block the
phosphorylation of EGFR and do not affect the phos-
phorylation of Her-2 while tyrphostin AG879 selec-
tively blocks the phosphorylation of Her-2 but not
EGFR activation. In addition, we tested emodin, an
inhibitor of both Her-2 and CK-2 [20,21]. As shown in
Figure 1A, in PC-3 and LNCaP cells the inhibitors of
EGFR, AG1478 and AG1517, diminished the phospho-
rylation of Akt. The inhibition of PI3K by the AG1517
inhibitor was dose-dependent (Fig. 1B). The specificity
of the Akt phosphorylation observed was confirmed by
using LY294002, a specific inhibitor of PI3K, the kinase
responsible of Akt phosphorylation (Fig. 1A, second
lane). LY294002 completely abrogated the phospho-
rylation of Akt. In contrast, in PC-3 and LNCaP cells,
Her-2 inhibitors (AG879 and emodin) showed no effect
on constitutive Akt phosphorylation (Fig. 1A), even
with high doses of inhibitor (Fig. 1B), suggesting that
Her-2 is not involved in the constitutive activation of
PI3K in prostate cancer cell lines. Together, these
observations indicate that Akt activation is strongly
dependent of EGFR signaling in prostate cancer cells.
To confirm the role of EGFR in PI3K/Akt activation
of prostate cancer cells and to eliminate the role of
a potential side effect of AG1478 and AG1517, we also
tested by transient transfection the effect of HERCD533
and mut-mycHer-2, two dominant mutant forms of
EGFR and Her-2 respectively. These truncated recep-
tors lack the cytoplasmic domain containing the kinase
activity and reduce the tyrosine phosphorylation of
their respective endogenous receptor [18,22]. LNCaP
308 Page et al.

cells were chosen as a model in this assay as these cells,
in comparison to PC-3 cells, were readily transfectable
and provided the more appropriate model to monitor
transfected protein expression by western-blot analy-
sis. In addition, the transfection control for HERCD533
was assessed by its ability to inhibit the activation of
endogenous EGFR receptor as no antibody are com-
mercially available for the detection by Western blot-
ting of the EGFR N-terminal sites. As shown in
Figure 1C, cells transfected with HERCD533 showed
a decreased phosphorylation of EGFR and Akt com-
pared to cells transfected with the empty vector
PCDNA3.1 or PCDNA3.1-mut-mycHer-2 (Fig. 1D).
These results again support the notion that EGFR, but
not Her-2, activates NF-kB through the PI3K/Akt sig-
naling pathway in prostate cancer cells.
The PI3K/Akt Pathway is Involved in the
Phosphorylation of IjBaand the Nuclear
Translocation of p65 in PC-3 Cells
PI3K has been involved in the constitutive activation
of NF-kB in PC-3 cells however the precise role of PI3K
in these cells is controversial. In particular, while two
reports cite a reduced NF-kB DNA binding in presence
of PI3K inhibitors [5,23] it has also been reported that
PI3K does not affect the nuclear localization of NF-kB
but rather the transcriptional ability of p65 subunit [6].
We used LY294002, a specific inhibitor of PI3K, to dis-
criminate the role of PI3K in the constitutive activation
of NF-kB in our PC-3 cells. As shown in Figure 2A and
B, LY294002 strongly decreased the transactivational
ability as well as the DNA binding of NF-kB in these
cells. As expected, no effect was observed in LNCaP
cells that do not present constitutive activation of NF-
kB. Furthermore, in PC-3 LY294002 prevented the
phosphorylation of IkBaon serines 32/36 and the
nuclear translocation of the p65 subunit of NF-kB
(Fig. 2C) confirming the role of Akt in the constitutive
activation of NF-kB in PC-3 prostate cancer cell line.
Constitutive Activation of CK-2
in Prostate Cancer Cells
As Her-2 does not appear to signal through the
PI3K/Akt pathway that leads to the phosphorylation of
IkBaon serines 32–36, we addressed which pathway
may be the downstream target of Her-2 and involved in
the constitutive activation of NF-kB in PC-3 cells. We
have previously shown that Her-2 activates NF-kB
through a mechanism that does not involve the phos-
phorylation of IkBaon serine 32–36 [11]. Interestingly,
in other tumor cell types, CK-2 has also been involved
in the constitutive activation of NF-kB by a mechanism
independent of IkBaphosphorylation on serine 32 – 36
[13–16]. CK-2 is constitutively activated in hormone-
insensitive PC-3 cells and to a lower extent in LNCaP
cells as measured by specific peptide phosphorylation
(Fig. 3A and [24,25]). This activity correlated with
the level of the catalytic subunits CK-2aand CK-2a0
(Fig. 3B) and was enhanced by the transient transfec-
tion of a wild type subunit CK-2a0construct (Fig. 3C)
while it was decreased by two CK-2 inhibitors, 4,5,6,7-
tetrabromobenzotriazole (TBB) and apigenin. We chose
these pharmacologic inhibitors since TBB is the most
Fig. 1. Activation of PI3K pathway by EGFR. A:InhibitionofPI3K
activation by EGFR inhibitors. PI3K activity was tested based on its
ability to phosphorylate Akt on serine 473. Thirty micrograms o f
total extracts from PC-3 and LNCaP cells treated for 6 hr with
DMSO (con trol) or tyrpho stin inhibitors. Cell extrac ts were loade d
on a 10% SDS PAGE and subjected to Western-blotting using anti-
phospho-serine473-Akt or anti-Akt antibodies. In all experiments
concentration of tyrphostininhibitors were 20 mMLY294002(LY),
40 mMemodin,10mMAG879,10mM AG1517, 250 nM AG1478, or
10 mMAG1296.B: Dosedependentinhibition of PI3Kby AG1517.Fifty
micrograms of totalextractsfromLNCaPcells treated for6 hr with
DMSO(control)orincreasingconcentrationof tyrphostin inhibitors
wereloadedona10%SDS ^PAGEandsubjectedtoWestern-blotting
with anti-phospho ser473-Akt, anti-Her-2 (control) or anti-Akt-1
(not shown) antibodies. Concentrations of tyrphostin inhibitors
were em odin 20, 4 0, 8 0 mM(lanes 2,6,and10 respectively), AG879
5,10, 20 mM(lane s 4,8,12 respectively),AG15172,10,20mM(lanes 3,7,
11 respectively).Inhibition of PI3K by dominant negative mutant of
EGFR but notby a dominant negative mutant of Her-2. LNCaPcells
were transfected with PCDNA3.1, HERCD533 a dominant negative
mut ant o f EGFR, (C) or a domina nt neg ative of Her- 2, Mut-Her- 2 (D).
After 4 8 hr, 50 mg of total extracts were subjectedto Western-blot-
tingusing an antibody recognizingthe transfectedconstructs (panel
C: anti-myc and panel D: anti-phosphoEGFR) or anti-phospho-
ser273Aktor anti-Akt1 antibodies.
Mechanism of NF-kB Activation in Prostate Cancer Cells 30 9

specific and selective CK-2 inhibitor [26,27] whereas,
though less specific, apigenin is more commonly used
as CK-2 inhibitor. Altogether these results show that
CK-2 is constitutively activated in PC-3 prostate cancer
cells line and to a lesser extent in LNCaP cells.
Role of CK-2 in the Constitutive
Activation of NF-jB
To ascertain a role of CK-2 in the constitutive
activation of NF-kB in PC-3 cells we used known in-
hibitors of CK-2, TBB, and apigenin, over a time period
that does not affect the phosphorylation status or the
expression of Her-2 (data not shown). Both inhibitors
significantly decreased the constitutive transactiva-
tional ability of NF-kB (Fig. 4A) in a dose dependent
manner (Fig. 4B) while the overexpression of CK-2a0
was correlated with this transcriptional activity. Inter-
estingly, the phosphorylation of IkB-aon serine 32/36
and the nuclear translocation of p65 was neither
affected by apigenin nor by TBB (Fig. 4C) supporting
the fact that Her-2 may activate the transactivational
ability of NF-kB through CK-2 activation in PC-3 cells.
Altogether, these results show that Her-2 activates NF-
kB, at least in part, through the activation of CK-2 in a
process independent of the phosphorylation of IkBaon
serines 32/36 and p65 nuclear translocation.
Her-2 Activates CK-2 in Prostate Cancer Cells
To determine whether EGFR and/or Her-2 are in-
volved in the constitutive activation of CK-2 in prostate
cancer cells, we tested the effect of tyrphostin inhibitors
on the CK-2 activity in PC-3 and LNCaP cells (Fig. 5A).
Fig. 2. Activation of NF-kB by PI3Kpathway. A:InhibitionofNF-
kB byinhibitors of PI3K.PC- 3 andLNCaPcells were co-transfected
with th e 3kB -c onA-luc a nd Renilla ve ctor s. Sixte en hour s followi ng
transfection, cells wereincubated for 24 hr with DMSOor 20 mMof
LY29004(LY).Cellswerethenassayedfor luciferase activities.Rela-
tive firefly luciferase activity (expressedin % control) is theratio of
luciferase activity in treated cells to that in control cells (DMSO).
Transfectionefficiency was normalizedto that of Renillaluciferase.
Values represent the mean SEM. *P<0.05 versus control (Stu-
dent’s t-test). B:InhibitionofNF-kB binding activity by PI3K inhibi-
tors. PC-3 and LNCaP cells were incubated for 6 hr with 20 mM
LY2940 02 (LY) and nuclear extracts were prepared and tested for
NF-kB DNAbinding activity a s describedin Material and Methods.
ns: not specific. C: Inhibition of p65 nuclear localization and IkBa
phosphorylation by PI3K inhibitor. Fifty micrograms of nuclear
extracts or cytoplasmic extracts from PC-3 cells treated for 6 hr
with DMSO (con trol) or 2 0 mM of LY294002 were loaded on a10%
SDS ^PAGE and subjected to Western-blotting using an anti-phos-
phoIkBa,anti-IkBa, anti-p65 or anti-Ran (a loading control for
nuclearextract) antibodies.
Fig. 3. A: Activity of CK-2 in prostate cancer cell lines. CK-2
activity was tested with10 mg of totalprotein and the activity was
determined as described in Materialand Methods. B:Westernblot
analysisofCK-2aandCK-2a.Celllysatesweresubjected toWestern
blotting and revealed using CK2 subunit specific antibodies.C:Inhi-
bition of CK-2 activity by apigenin and TBB.PC-3 cells were stimu-
lated for 6 hr with 50 mMapigeninor25mM of TBB or transfected
for 24 hr with empty ve ctor or CK-2awild type construction.CK-2
activity was tested with10 mg of totalprotein and the activity was
determinedas described (Materials and Methods).Values (panels A
and C) represent themean SEM. Significance, as c ompared to the
control, was defined as *P<0.05 using a S tuden t’s t-test.
310 Page et al.

In PC-3 cells, the Her-2 inhibitor AG879 decreased the
CK-2 activation by 75% while EGFR inhibitors AG1517
had a weaker inhibitory effect. As AG1478 had no
significant effect, this would argue against a role of
EGFR in constitutive activation of CK-2 in PC-3 cells.
Similarly, in LNCaP cells only AG879 showed a signi-
ficant effect on the constitutive activation of CK-2. This
inhibition was weaker than in PC-3 cells and may be
related the high expression of Her-2 in LNCaP cells
compared to PC-3 cells [11]. We verified that the inhi-
bitory effect of tyrphostin inhibitors on CK-2 activation
was not due to a downregulation of the CK-2 protein
expression (Fig. 5B), supporting the notion that Her-2 is
involved in the activation of CK-2. To confirm this
result we also tested the effect of mut-mycHer-2 ex-
pression by transient transfection assays. As shown in
Figure 5C, mut-mycHer-2 almost completely abroga-
ted CK-2 activity, again supporting the idea that Her-2
is involved in the constitutive activation of CK-2 in
prostate cancer cells.
Fig. 4. Activation of NF-kBbyCK-2kinase.A: PC-3 cells were
co-transfectedwithRenilla and3kB-conA-luc vectorsanda control
or a CK-2a0encoding vector. Sixteenhours after transfection, cells
were incubatedin media with either DMSO, an appropriate control,
50 mM apigenin (Api) or 25 mM TBB for 24 hr.Cells were then as sayed
for luciferase activity. Relative firefly luciferase ac tivity (expressed
in % control) is theratio of luciferase activity in treated cells to that
in control cells (DMSO or control vector). Values represent the
mean SEM. *P<0.05 versus control(Student’st-test).B:PC-3cells
were co-transfected with the Renilla and 3kB-conA-luc vectors.
Sixteenhours after transfection, cells were incubatedin media with
either DMSO, or indicated doses of apigenin (Api) orTBB for 24 hr.
Cellswere then assayed forluciferaseac tivity.Relativefireflylucifer-
ase activity(expressedin % control) is theratio of luciferase activity
in treated cells to that in control cells (DMSO or control vector).
Values represent the mean SEM. *P<0.05, **P<0.10 versus con-
trol (Stude nt’s t-test).C:50mgof nuclear extracts (NE) or cytoplas-
micextracts (CE)fromPC-3 cells treatedfor6 hr witheitherDMSO,
50 mM apigenin, or 25 mM TBB wereloaded ona 10% SDS ^PAGEand
subjec ted to Western blotting using either an anti-p65, anti-phos-
phoserines 32 and36 IkBaoranti-IkBaantibodies. Anti-Ranserved
as theloading controlfor nuclear extracts.
Fig. 5. Constitutive activation of CK-2 by Her-2. A:Inhibitionof
CK- 2 ac tivity by tyrphos tin.Pros tate ca ncer cells were treated with
tyrphos tin inhibitor s and 10 mg of totalcell extracts were tested for
CK-2 activity.Relative CK-2 activity(expressedas %) was calculated
as theratio of CK2ac tivity inDMSO-treated cells (control,10 0%)to
that in cells treated with tyrphostins inhibitors. Concentrations
were10 mMAG879,10mMAG1517,or250nMAG1478.B: Expression
of CK-2 inthe presence of tyrphostininhibitors.Fourtymicrograms
of total extract from PC-3 cells used in Figure 4Awere loaded on
SDS ^PAGE and subjected to Western-blotting using anti-CK-2a
anti-CK-2a0,actin(control)antibodies.C: InhibitionofCK-2activity
by Her-2 mutant.Cells were transfected with PCDNA3.1 or mut-
Her-2 and 24 hr later 2.5 mg of total extracts were tested for CK-2
activity.Relative CK-2 activity(expresseda s %)is the ratios of CK- 2
activity in control PCDNA3.1 cells (100%) to thatin mut-Her-2 cells.
Values (panels A and B) represent themean SEM. Significance, as
compared to the control, was defined as *P<0.05 using a Student
t-test.
Mechanism of NF-kB Activation in Prostate Cancer Cells 311

DISCUSSION
In prostate cancer cells, the sequential signaling
events involved in the constitutive activation of NF-kB
are not clearly defined. It has previously been shown
that in hormone-independent PC-3 cells EGFR and
Her-2 induce the activation of NF-kB through two
distinct mechanisms [11]. We have previously demons-
trated using selective tyrphostin inhibitors that EGFR
activates NF-kB through a mechanism involving the
phosphorylation of IkBaon serine 32/36 thereby alte-
ring the nuclear translocation of p65 [11]. In contrast,
Her-2 activates a signaling pathway that neither affects
the nuclear translocation of p65 nor the phosphoryla-
tion of IkBaon N-terminal serines 32/36 [11]. Here we
extend these observation to show that in PC-3 cells,
EGFR, but not Her-2, is involved in the constitutive
activation of the PI3K/Akt signaling pathway. Inhibi-
tion of the PI3K/Akt signaling pathway leads to a
decreased phosphorylation of IkBaon serines 32 – 36
and the nuclear translocation of p65. The similarity of
effects between EGFR and PI3K/Akt inhibitors, on the
constitutive NF-kB activation in PC-3 cells, strongly
suggests a model where EGFR is involved in the acti-
vation of NF-kB through the PI3K/Akt activation
(Fig. 6). Furthermore, in line with our results obtained
in PC-3 prostate cancer cells, PI3K has also been impli-
cated in the constitutive activation of NF-kB in several
cancer cell type through stimulation by various growth
factors, including ErbB ligands [8–10].
In addition, here we demonstrate for the first time
that Her-2 also participates in the constitutive NF-kB
activation through the activation of CK-2. CK-2 has
previously been identified as a kinase responsible for
the constitutive phosphorylation of C-terminal sites of
IkBa[13–15,28,29]. This phosphorylation regulates its
intrinsic stability but does not affect its stimulated
degradation [28]. Moreover, the inhibition of CK-2 acti-
vation does not substantially impair either the degra-
dation of IkBaor the nuclear translocation of the p65
subunit of NF-kB [28,30]. Similarly, we have demons-
trated that in PC-3 cells, inhibition of CK-2 neither
impairs the phosphorylation of IkBaon serine 32/36
nor the nuclear translocation of the p65 subunit of NF-
kB. In addition we also observed that Her-2 is involved
in the constitutive activation of CK-2 in PC-3 cells, and
this provides an explanation for the observation that
Her-2 signaling does not affect the phosphorylation
of IkBaon serine 32/36 (Le Page et al. in press).
Altogether, these results strongly implicate Her-2 in the
constitutive activation of NF-kB through the activation
of CK-2 (Fig. 6). This is the first direct evidence show-
ing CK-2 as a component of the constitutive signaling
mediated by Her-2. It would be interesting to extend
this observation to other epithelial cancer cell type
presenting a constitutive activation of Her-2 such as
breast cancer cells.
Surprisingly, in LNCaP cells the overexpression and
activation of Her-2 [18,19] (Le Page et al. in press), as
well as the constititutive activation of PI3K [5,6,23]
(Fig. 1), is not sufficient for inducing a constitutive NF-
kB activation [1,5,6,23]. Several studies have attempted
to explain the reason for the lack of constitutive NF-kB
activity in LNCaP cells. One possibility is that this
phenomenon is due to the presence of the androgen
receptor (AR). Indeed, the absence of androgen in the
culture media of LNCaP cells induces NF-kB activa-
tion, while addition of androgen inhibits this activation
[24,31,32]. The precise mechanism of such inhibition is
still unknown, although protein interaction between
p65 and AR as well as AR and EGFR have been pro-
posed to be responsible for this interference [33,34]. In
addition, the expression of REPS2, a recently described
protein expressed in LNCaP cells, has also been shown
Fig. 6. Model representing mechanisms of constitutive NF-kB
activation in prostate ca ncer cells. In prostate ca ncer cells, we pro-
pose that EGFRac tivates the PI3K/Aktpathway leading to IKK acti-
vation, phosphorylation of IkBaon serine 32/36 and subsequent
nuclear translocation of NF-kB. Her-2 signaling through CK-2
enhances the transactivationalabilityof NF-kB. Additional signaling
mediators such as MAPK and NIK have alsobeen shown to partici-
pa te to the c ons titutive NF-kB activation[4].Severalpharmacologic
and endogenous inhibitors can interfere at each step of this
activation.
312 P age et al .

to act as an inhibitor of p65 [34] and may also explain
the lack of NF-kB activation in LNCaP cells.
In prostate cancer patients, overexpression of EGFR
and Her-2 as well as activation of Akt, CK-2, and NF-kB
correlates with poor prognosis and/or high Gleason
score [35–41]. Consequently inhibition of both EGFR
and Her-2 may be an interesting therapeutic strategy.
Indeed the presence of these two independent activa-
tion pathways suggests that strategies to inhibit NF-kB
in prostate cancer cells through selective inhibition of
one receptor may not be completely efficient. In addi-
tion, our study does not exclude the involvement of
other active components of such as MAPK and NIK,
which have already been shown to be constitutively
activated in prostate cancer cell lines [42–46] and
potentially involved in the constitutive activation of
NF-kB in these cells [4]. Interventions that target
intracellular proteins, in conjunction with receptor in-
hibitors, are promising but require the precise iden-
tification of such signaling pathway in order to act
optimally. One of the lessons learned with this class of
targets is that we currently do not know how to opti-
mally apply them to the treatment of cancer due to the
lack of knowledge about the specific role of each ErbB
and their signaling in particular cancers. For example,
in contrast to the mechanism observed in breast cancer
cell lines [47], Her-2 is not involved in the activation of
PI3K and CK-2 is not implicated in IkBadegradation or
p65 translocation in PC-3 prostate cancer cells (Figs. 1
and 4C). In addition, negative effects on normal cells
with more closely matched receptor binding capacities
may bring dramatic side effects. Interventions that
target the intracellular protein, in conjunction with a
receptor inhibitor may be more promising. Here we
have demonstrated the additional implication of Akt
and CK-2 as independent signaling pathways consti-
tutively activated by EGFR and Her-2 respectively.
As their constitutive activities revealed here appear
to be present in the native cell lines without the need
of genetic manipulation associated with transfection
studies, this suggests that they are biologically relevant
and may more closely represent what occurs during
disease progression.
CONCLUSION
In conclusion, this study improves our understan-
ding of the ErbB-mediated signaling and shows for the
first time CK-2 as a specific component of Her-2 sig-
naling. We also demonstrate the independent implica-
tion of PI3K and CK-2 in the constitutive activation NF-
kB by EGFR and Her-2 respectively. This allows us to
propose a more complete model about the mechanism
of constitutive activation of NF-kB in prostate cancer
cells (Fig. 6) which appears different from the mechan-
ism previously observed in cancer cell lines such as
breast cancer, where EGFR and Her-2 act as hetero-
dimers. It will be important to extend the present
observations to cancer tissues, in the presence of thera-
peutics that target ErbB members, to determine the
extent to which these signaling pathways are modu-
lated in prostate cancer patients. In this way it will be
possible to determine whether the dual activation of
NF-kB through both EGFR and Her-2 is an important
consideration when attempting to devise novel anti-
cancer therapies for androgen-independent and/or re-
fractory prostate cancer.
ACKNOWLEDGMENT S
The authors thank members of Institute of Cancer of
Montreal for helpful discussion and technical support.
We particularly thank Nathalie Delvoye for technical
assistance and Jean-Simon Diallo for critical reading of
the manuscript. We are very grateful to Dr Ming-Fong
Lin for providing Her-2 and mut-Her-2 plasmids, to
Dr Sylvain Meloche and Dr Alex Ullrich for HERCD533
construction and to Dr. Lichfield for CK-2a0construc-
tion. We also thank Dr Juana Wietzerbin for providing
ConA-luc and kB-ConALuc vector.
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