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RESEARCH PAPER
Osteoblast-induced EGFR/ERBB2 signaling in androgen-sensitive
prostate carcinoma cells characterized by multiplex kinase
activity profiling
A
˚
se Bratland Æ Piet J. Boender Æ Hanne K. Høifødt Æ Ingrid H. G. Østensen Æ
Rob Ruijtenbeek Æ Meng-yu Wang Æ Jens P. Berg Æ Wolfgang Lilleby Æ
Øystein Fodstad Æ Anne Hansen Ree
Received: 4 June 2008 / Accepted: 27 February 2009 / Published online: 18 March 2009
Ó The Author(s) 2009. This article is published with open access at Springerlink.com
Abstract Bone metastases in prostate cancer are pre-
dominantly osteoblastic. To study regulatory mechanisms
underlying the establishment of prostate cancer within an
osteoblastic microenvironment, human androgen-sensitive
prostate carcinoma cells (LNCaP) were treated with culture
medium conditioned by human osteoblast-derived sarcoma
cells (OHS), and activated signalling pathways in the car-
cinoma cells were analyzed using microarrays with
tyrosine kinase substrates. Network interaction analysis of
substrates with significantly increased phosphorylation
levels revealed that signalling pathways mediated by
EGFR and ERBB2 were activated in LNCaP cells under
OHS influence but also by androgen treatment. Activation
of EGFR/ERBB2 signalling was also found in LNCaP cells
in cocultures with OHS cells or osteoblastic cells that had
been differentiated from human mesenchymal stem cells.
Our experimental data suggests osteoblast-directed induc-
tion of signalling activity via EGFR and ERBB2 in prostate
carcinoma cells and may provide a rationale for the use of
EGFR or ERBB2 inhibition in systemic prevention or
treatment of metastatic prostate cancer in the androgen-
sensitive stage of the disease.
Keywords Androgen EGFR ERBB2 Kinase array
Metastasis Osteoblast Prostate carcinoma
Abbreviations
FBS Fetal bovine serum
EGF Epidermal growth factor
Introduction
Metastatic prostate cancer is a leading cause of cancer
morbidity and mortality. The skeleton is the principal
organ for metastasis formation in prostate cancer, and bone
metastases are frequently painful and debilitating. Whilst
androgens are critical regulators of prostate carcinoma
growth and progression, most patients respond only
temporarily to androgen ablation therapy. Skeletal metas-
tases are predominantly osteoblastic, as apparent both
A
˚
. Bratland H. K. Høifødt M.-y. Wang Ø. Fodstad
A. H. Ree (&)
Department of Tumor Biology, Oslo University Hospital,
Montebello, 0310 Oslo, Norway
e-mail: a.h.ree@medisin.uio.no
A
˚
. Bratland W. Lilleby
Division of Cancer Medicine and Radiotherapy,
Oslo University Hospital, Oslo, Norway
P. J. Boender R. Ruijtenbeek
PamGene International BV, ‘s-Hertogenbosch, The Netherlands
I. H. G. Østensen
Norwegian Microarray Consortium, Oslo University Hospital,
Oslo, Norway
I. H. G. Østensen
Department of Molecular Biosciences, University of Oslo,
Oslo, Norway
J. P. Berg
Faculty Division Ulleva
˚
l University Hospital,
University of Oslo, Oslo, Norway
A
˚
. Bratland Ø. Fodstad
Faculty Division The Norwegian Radium Hospital,
University of Oslo, Oslo, Norway
A. H. Ree
Faculty Division Akershus University Hospital,
University of Oslo, Oslo, Norway
123
Clin Exp Metastasis (2009) 26:485–496
DOI 10.1007/s10585-009-9248-9
radiographically and histopathologically, and are associ-
ated with elevated serum level of bone-specific alkaline
phosphatase, a marker of osteoblast proliferation [1, 2].
These observations suggest that the biological interaction
between prostate carcinoma cells and osteoblasts contrib-
utes to the metastatic progression of prostate cancer.
In order to experimentally address how osteoblastic cells
may influence prostate carcinoma cell biology upon for-
mation of bone metastasis, the human, androgen-sensitive
prostate carcinoma cell line LNCaP [3] was treated with
culture medium conditioned by the human, osteoblast-
derived sarcoma cell line OHS [4], and subsequent multi-
plex profiling of LNCaP kinase activity was performed
using flow-through microarrays with peptide substrates
(Tyrosine Kinase PamChip
Ò
Arrays; PamGene Interna-
tional BV, ‘s-Hertogenbosch, The Netherlands), a novel
platform that allows rapid, real-time measurements of
phosphopeptide signatures generated by biological sam-
ples. As supplementary approaches, we used LNCaP cells
in cocultures with either OHS cells or with osteoblastic
cells that had been differentiated from human mesenchy-
mal stem cells.
Insight into regulatory mechanisms underlying the
establishment of prostate carcinoma cells within an osteo-
blastic microenvironment may eventually lead to more
effective therapies to prevent or treat metastatic disease.
Hence, from a therapeutic perspective, we compared the
intracellular LNCaP signalling pathways activated by
influence of osteoblastic cells with pathways induced by
androgen treatment, to identify signalling networks
potentially accessible for therapeutic targeting.
Materials and methods
Cell cultures
The LNCaP and OHS cell lines were routinely held in
RPMI 1640 medium supplemented with 10% fetal bovine
serum (FBS) and 2.0 mM glutamine, defined as growth
medium. Seventy-two hours before start of experimental
incubations, monocultures of LNCaP and OHS were see-
ded in a total number of 1.0 9 10
6
cells in 75 cm
2
cell
flasks in RPMI containing 2% charcoal-treated FBS and
glutamine. After 48 h, this medium was changed to RPMI
containing 0.5% charcoal-treated FBS and glutamine,
defined as experimental medium, for another 24 h before
experimental incubations were started (at time 0). All cell
cultures were invariably held in 10 ml medium throughout
different incubations.
At time 0 (start of the experimental incubations),
LNCaP cells were refed with experimental medium sup-
plemented with 100 nM of the synthetic androgen analog
R1881 (methyltrienolone; Biocompare, Inc., South San
Francisco, CA), giving rise to the LNCaP entity denoted
androgenic, or with medium conditioned by OHS cells.
The conditioned medium had been collected from OHS
monocultures that had been grown for 48 h (relative to
time 0) in experimental medium. This medium was sub-
sequently diluted 1:10 in either fresh experimental medium
or in medium obtained from standard LNCaP monocultures
after 48 h of incubation (relative to time 0) in experimental
medium, before the application onto monocultured LNCaP
cells, giving rise to the LNCaP entities denoted paracrine 1
and paracrine 2, respectively.
Cocultures of LNCaP/OHS were also seeded in a total
number of 1.0 9 10
6
cells in 75 cm
2
cell flasks in 10 ml
medium and were incubated identically to the monocul-
tured cell lines prior to and during experimental
incubations. Different ratios of LNCaP to OHS cells had
been tested in a series of cocultures to find the optimal
culturing conditions [5], and a 10:1 seeding ratio was
chosen.
Differentiated mesenchymal stem cells from human
bone marrow were prepared as previously described [6, 7].
Briefly, bone marrow (10–20 ml) was aspirated from the
posterior iliac crest of healthy, adult volunteers. Mononu-
clear cells were isolated by density gradient centrifugation
and resuspended in complete medium, consisting of MEMa
supplemented with 20% FBS and 2.0 mM glutamine. After
24 h, non-adherent cells were discarded, and the adherent
cells were thoroughly washed and further cultured in
complete medium for 7 days. Subsequently, the cells were
detached and replated in osteogenic differentiation med-
ium, consisting of MEMa with 10% FBS, 1.0 mM
glutamine, 10 nM dexamethasone, 10 lM b-glycerol
phosphate, and 200 lM ascorbic acid. This medium was
replaced every 3–4 days for about 20 days before LNCaP
cells (1.0 9 10
6
cells in 75 cm
2
cell flasks) were seeded on
top of the osteoblasts and the cocultures of LNCaP and
differentiated stem cells were incubated identically to the
other culture setups prior to and during experimental
incubations.
In the experimental setups, LNCaP cells were harvested
48 h after time 0, either directly from the various mono-
cultures or after immunomagnetic cell segregation of
cocultures (see below), and lysed in M-PER Mammalian
Extraction Reagent containing Halt Phosphatase Inhibitor
Cocktail and EDTA-free Halt Protease Inhibitor Cocktail
(Pierce Biotechnology, Inc., Rockford, IL). Reference
lysates were made from monocultured LNCaP grown for
48 h (relative to time 0) in experimental medium only.
In the experimental setup involving epidermal growth
factor (EGF; Sigma–Aldrich Norway AS, Oslo, Norway),
EGF was added to LNCaP cells in a final concentration of
50 ng/ml.
486 Clin Exp Metastasis (2009) 26:485–496
123
Kinase activity profiling
The kinase substrate array technology allows functional
comparison of biological samples without prior knowledge
of which activity pathways are influenced by the experi-
mental conditions. The high-throughput format of the
Tyrosine Kinase PamChip
Ò
Array is based on the use of a
porous, three-dimensional aluminum-oxide material as
solid support for the substrates. The sample lysates are
actively pumped through the interconnected capillary pores
of the arrays to allow contact with the reactive surface for
enzymatic reaction with the peptide substrates. The phos-
phorylation kinetics are therefore rapid and can be
completed within few minutes, allowing the generation of
spot images to be followed in real-time.
Each array contains 144 peptide substrates, and these
target peptides consist of 13 or 14 amino acids with sites
for phosphorylation, mainly tyrosine, representing 100
different proteins. Reaction mixtures consisted of Abl
Reaction Buffer (50 mM Tris–HCl pH 7.5, 10 mM MgCl
2
,
1 mM EGTA, 2 mM dithiothreitol, 0.01% Brij 35; New
England BioLabs, Inc., Ipswich, MA), 1 mg/ml bovine
serum albumin, 100 lM ATP, and 12.5 lg/ml of the
monoclonal, FITC-conjugated anti-phosphotyrosine anti-
body (Exalpha Biologicals, Inc., Maynard, MA), added to
each sample lysate, containing 2–5 lg total protein. The
arrays were blocked with 20 lg/ml bovine serum albumin
and subsequently washed twice with Abl Reaction Buffer
before the reaction mixtures were applied for initiation of
enzymatic reactions. Spot images were recorded after each
completed pumping cycle by a charge-coupled device
camera until the reactions were terminated.
Raw and processed data and investigation and array
designs can be obtained from the EMBL-EBI ArrayExpress
database (www.ebi.ac.uk) by accession number E-TABM-
626.
Microarray data adaptation and statistical analysis
Six independent LNCaP reference samples (denoted
LNCaP baseline), one of each LNCaP treated sample
(paracrine 1, paracrine 2, androgenic), one sample each of
LNCaP and OHS cells immunosegregated from coculture
(see below), and two independent samples of monocultured
OHS cells were analyzed, and each sample was measured
in three independent reactions. The image information was
converted using BioNavigator software (PamGene Inter-
national BV, www.pamgene.com). For each spot on the
array, signal intensity after background subtraction was
calculated and used for further analysis. Data normalization
of spot signal intensities and subsequent comparison
analyses were conducted using GeneSpring software
(Agilent Technologies, www.home.agilent.com). All values
were normalized to the calculated mean value of all sub-
strate phosphorylation intensities in the LNCaP baseline
sample. In the LNCaP treated samples, the criterion for
selecting a substrate with significantly increased phos-
phorylation level compared to the corresponding value in
the baseline sample was set at a statistical difference of
P \ 0.05 (Student’s t-test). Using this criterion, the fold-
increase (log
2
) of substrate phosphorylation levels was
within a range of 0.85 to [ 5.2.
Following the statistical analysis, two different pathway
visualization systems were used to create information about
pathway connectivity. Peptide names were generated from
corresponding gene name entries and protein sequences in
SwissProt (http://au.expasy.org/sprot) to enable unambigu-
ous annotation of the peptide sequences and phosphorylation
sites. It should be noted that, for some proteins, multiple
peptides have been derived and are present on the Tyrosine
Kinase PamChip
Ò
Array. For the pathway connectivity
analysis, one list of peptide identifications for each of the
treated samples was generated. First, those lists were loaded
into PathwayArchitect software (Stratagene Corp.,
www.stratagene.com; Strand Life Sciences Pvt. Ltd.,
www.avadis.strandgenomics.com), and the program was
used to generate a new list that contained all peptides in the
others, but where duplicates had been removed. The peptide
identifications in the final list were visualized through a
direct interaction network, defined to show all interactions
between peptides that were of the following types: binding,
expression, protein modification, and regulation. Some
peptides were omitted from the network because the inter-
actions were below maximum score/quality, which is
defined as an interaction from manually curated sources in
the database. The direct interaction network was exported,
all interactions were manually checked, and uninteresting
interactions were removed. These manual edits were incor-
porated into the interaction network. Subsequently, the
peptide list identified for each of the treated samples was
imported into PathwayStudio software (Ariadne Genomics,
www.ariadnegenomics.com), and pathways were created
directly from the ResNet database (Ariadne Genomics),
which comprises both curated and canonical pathways. The
peptide set was cross-checked with these pathways, and a
pathway list was generated and ranked by the hypergeo-
metric probability factor. All interactions were manually
inspected and selected on the criterion of the highest number
of proteins being involved in a linear pathway or sub-path-
way, by superposition of the peptide set to a cartoon
representation of the pathway reported.
Western blot analysis
Expression of phosphoproteins was measured by means of
standard western immunoblots. Primary, polyclonal
Clin Exp Metastasis (2009) 26:485–496 487
123
antibodies, selected against the same phosphorylated epi-
topes as the corresponding target peptides on the
microarrays, were anti-phospho(y1197)-EGFR (Abcam
Plc., Cambridge, UK) and anti-phospho(y1248)-Neu (Santa
Cruz Biotechnology, Inc., Santa Cruz, CA). Further, the
monoclonal anti-EGFR antibody 425.3 [8], a polyclonal
anti-ErbB2 antibody (R&D Systems Europe Ltd., Abing-
don, UK), and a monoclonal anti-a-tubulin antibody
(Calbiochem/Merck Biosciences Ltd., Nottingham, UK)
were used.
Immunomagnetic cell separation
MOC-31 (IQ Corporation BV, Groningen, The Nether-
lands) is an IgG1 class antibody that binds to the EPCAM
antigen, which is consistently expressed in most epithelial
cells [9]. The high-affinity monoclonal antibody 9.2.27,
which was originally developed against melanoma [10],
recognizes an epitope on the high molecular weight mel-
anoma-associated antigen and has also been shown to bind
some subgroups of sarcoma, including osteosarcoma [11,
12]. The antibodies were conjugated to superparamagnetic
particles coated with polyclonal sheep-antimouse IgG
particles (Dynabeads; Dynal A.S., Oslo, Norway).
At the end of experimental incubations, cocultures of
LNCaP cells with OHS cells and of LNCaP cells with
differentiated mesenchymal stem cells were detached, and
the resulting single cell suspensions were subjected to
immunomagnetic target cell isolation, essentially as pre-
viously described [12–15]. The positive cell fractions were
examined by light microscopy for the principal presence of
cells with C5 immunobeads bound to their surface (bead
rosettes). For the purpose of cell quantification and quality
assurance, the immunosegregation procedure was repeated
with 9.2.27-coated beads added to the negative cell frac-
tions for isolation of OHS cells. The segregated cell
fractions from LNCaP/OHS coculture samples (10
6
–10
7
cells) contained 60–70% MOC-31-selected bead rosettes
and *30% 9.2.27-selected bead rosettes.
Results
Kinase activity evoked by osteoblastic and androgenic
influence
Monocultured LNCaP cells were treated with OHS-con-
ditioned medium to experimentally replicate the biological
context of paracrine osteoblastic influence. Moreover, to
simulate the complex processes involved in aberrant acti-
vation of the androgen signalling axis in prostate cancer
[16, 17], the experimental setup included LNCaP cells
treated with the synthetic androgen analog R1881 to
observe whether androgen receptor-mediated signalling
pathways might differ from pathways activated by OHS-
directed influence. Given the assumption that biologically
relevant signalling events implicated in the metastatic
phenotype require sustained activation, 48-h incubation
times were used for the experimental LNCaP contexts.
From the tyrosine kinase microarray analysis, the sub-
strate phosphorylation state generated by each LNCaP
entity (paracrine, androgenic) was calculated with respect
to baseline (untreated LNCaP cells). The identified sub-
strates showed increases in phosphorylation level within a
broad range (Table 1).
Each individual substrate phosphorylation signature was
considered to represent a subset of the information flow
through the globally activated signalling network of the
particular LNCaP entity. Tools for analyzing interconnec-
tivity of biological molecules have so far been used
primarily to explore system-level gene expression data.
However, such computational methods may also elucidate
how kinase activity information is directed, and by using
these algorithms, we assumed that phosphorylation events
that appeared simultaneously might be interlinked and
provide information about pathway connectivity [18, 19].
By applying these assumptions, the network interaction
analysis omitted phosphorylated substrates that did not
appear within any signalling pathway when defined by the
interaction types delineated in ‘‘Materials and methods’’ .
As illustrated in Fig. 1, the resulting network connectivity
map indicated that signalling pathways involved in cell
adhesion and motility as well as cell proliferation were
activated in the paracrine LNCaP entity. Activation of
similar but also completely unrelated proliferation path-
ways was observed in the androgenic entity. Interestingly,
only the signalling pathway mediated by EGFR seemed to
be activated by the influence of both osteoblastic cells and
androgen treatment.
Since the technology for multiplex kinase activity pro-
filing and computational tools for data analysis are at an
early stage of development [20], no general consensus
approach to data validation exists. We therefore performed
conventional western immunoblotting for selected, indi-
vidual phosphoproteins expressed by LNCaP cells [21, 22]
that in addition are representative of signalling pathways
and therapeutic targets under investigation in recently
conducted trials of metastatic prostate cancer [23–26].
Markedly increased expression levels of EGFR phosphor-
ylated on tyrosine 1197 and ERBB2 phosphorylated on
tyrosine 1248 were found in both the paracrine and
androgenic LNCaP entity (Fig. 2a). In contrast, EGFR was
found to be non-phosphorylated following 48 h of incu-
bation with either EGF or 10% FBS, whereas short-
time EGF treatment caused transient increase in EGFR
phosphorylation (Fig. 2b), which is in accordance with
488 Clin Exp Metastasis (2009) 26:485–496
123
previously published data [21, 27, 28] and may indicate
that functional signalling in prostate carcinoma cells under
influence of osteoblastic cells is diverse and complex [21].
EGFR/ERBB2 phosphorylation in supplementary
biological models
We used additional experimental setups as biological
controls for the osteoblastic influence on prostate carci-
noma cells; the first to model the direct interaction between
LNCaP and OHS cells and the second to provide cells that
might be more representative of physiological osteoblasts.
Culturing LNCaP cells with OHS cells caused sub-
stantial change in LNCaP morphology. The spindle-shaped
feature of monocultured LNCaP cells (Fig. 3a) was rapidly
lost upon direct contact with the OHS cells. In coculture,
both cell types appeared rounded, although cytoplasmic
processes were still apparent on LNCaP cells (Fig. 3b).
Cellular morphology of the OHS cells (Fig. 3c), however,
remained independent of the culturing conditions.
Non-hematopoietic stem cells in the bone marrow are
capable of differentiating into a variety of tissue entities,
including osteogenic cells of bone tissue [29]. Incubation
of mononuclear cells isolated from adult, human bone
marrow with mesenchymal stem cell-stimulating medium
followed by osteogenic differentiation medium [6, 7] gave
rise to cells with osteoblastic characteristics, for example
mineral deposition (Fig. 3d, e) and alkaline phosphatase-
secreting activity (not shown), to be used in coculture with
LNCaP cells (Fig. 3f).
Table 1 Peptide substrates with increased phosphorylation levels
generated by the paracrine and androgenic LNCaP entities
Peptide substrate Paracrine
a
Androgenic
EGFR_y1197
b
1.84
c
1.94
EGFR_y1110 2.34 –
RB1_s807/s811 3.39 3.07
ERBB2_y877 1.67 –
ERBB2_y1248 1.56 –
CDK2_t14/y15 1.86 –
EPHB1_y778 2.11 –
IRS2_y919 5.56 –
JAK1_y1022/y1023 2.05 –
LAT_y200 3.49 –
LCK_y394 1.91 –
MET_y1230/y1234/y1235 1.57 –
MST1R_y1353 2.86 –
MST1R_y1356/y1360 3.38 –
PDPK1_y9 1.82 –
PDPK1_y373/y376 1.82 –
PTK2_y576/y577 1.39 –
PTK2B_y579/y580 1.21 –
RASA1_y460 1.63 –
RET_y1029 1.76 –
ZAP70_y492/y493 1.93 –
CREB1_y134/s133 – 1.47
ERBB4_y1284 – 3.31
GSK3B_y216 – 1.15
RAF1_s337/s338/y339/y340 – 1.14
CHRNB1_y390 1.57 1.57
LTK_y772/y776/y777 – 1.20
DDR1_y792/y796/y797 1.20 –
MAPK10_t221/y223 1.32 –
MAPK12_t183/y185 – –
PECAM1_y713 2.06 –
PRRX2_y214 1.48 –
ANXA1_y20/t23 2.41 –
CD79A_y182/y188 1.75 –
CTTN1_y477/y483 2.01 –
CTTN1_y499 2.13 –
ENO2_y43 1.85 –
EPHA2_y772 1.99 –
EPHA7_y608/y614 2.27 –
EPOR_y368 2.08 –
EPOR_y426 2.29 –
FER_y714 1.66 –
FES_y713 2.20 –
FGFR2_y769 1.77 –
FGFR3_y760 2.23 –
FRK_y387 2.02 –
LAT_y255 1.54 –
Table 1 continued
Peptide substrate Paracrine
a
Androgenic
NTRK2_y702/y706/y707 1.43 –
PDGFRB_y579/y581 3.87 –
PDGFRB_y716 1.78 –
PIK3R1_y607/s608 2.09 –
PXN_y31 1.88 –
PXN_y118 2.07 –
TEC_y519 1.68 –
PFKFB1_s33 – 1.36
PTPN11_y542 – 2.94
SYN1_s9 – 1.20
‘–’ denotes that the change in substrate phosphorylation level was not
found to be significant (P \ 0.05, Student’s t-test)
a
Phosphopeptide values from the LNCaP paracrine 2 entity are given
b
For each substrate, position of phosphorylation sites within the
protein is indicated
c
Fold changes (log
2
) relative to LNCaP baseline samples are listed
Clin Exp Metastasis (2009) 26:485–496 489
123
We applied the immunomagnetic cell separation method
for selective isolation of LNCaP cells from the cocultured
osteoblastic cells (Fig. 3g) and subjected the isolated
carcinoma cells to western immunoblotting. Increased
expression levels of EGFR phosphorylated on tyrosine
1197 and ERBB2 phosphorylated on tyrosine 1248 were
found in LNCaP cells from coculture with both OHS cells
and osteoblastic cells that had been differentiated from
human mesenchymal stem cells (Fig. 2a).
Supplementary kinase activity profiling
To assess robustness of the biological models in the ana-
lytical application of multiplex kinase activity profiling,
three experimental setups were exploited.
Kinase activity was compared in LNCaP cells treated
with medium conditioned by OHS cells only and with the
addition of medium obtained from standard LNCaP
monocultures (LNCaP entities denoted paracrine 1 and
paracrine 2, respectively). In this experimental setting, the
profile of phosphorylated substrates generated by the
LNCaP cells did not significantly change by the addition of
medium conditioned by LNCaP monocultures (compare
Table 1 with Table 2), strongly indicating that the signal-
ling pathways of biological importance in the paracrine
setting were activated by factors secreted by the OHS cells
exclusively.
Kinase activity induced in LNCaP cells by direct contact
with OHS cells was analyzed following carcinoma cell
isolation from cocultures. The substrate phosphorylation
state generated by such LNCaP cells overlapped the
phosphopeptide signatures identified in the paracrine
LNCaP entities (Table 2).
ERBB4
EGFR
MST1R
PTK2B
LAT
ZAP70
RASA1
PTK2
PDPK1
EPHB1
CDK2
MET
RET
JAK1
IRS2
LCK
ERBB2
CREB1
GSK3B
RB1
RAF1
Fig. 1 Interconnected
signalling pathways activated in
LNCaP cells by influence of
osteoblastic cells or androgen
treatment. Two pathway
visualization systems were
applied to the data set (Table 1),
which resulted in almost
identical network connectivity
maps. The substrate annotations
are derived from gene name
entries in SwissProt. The lines
connecting nodes represent
interactions of the following
types: binding, expression,
protein modification, and
regulation. Yellow and blue
nodes symbolize substrates
phosphorylated by the paracrine
and androgenic LNCaP entities,
respectively. (Color figure
online)
α-tubulin
EGFR
p-EGFR
(170kDa)
b
a
12345
123456
α-tubulin
ERBB2
EGFR
p-ERBB2
(185 kDa)
p-EGFR
(170 kDa)
Fig. 2 Expression of phospho(y1197)-EGFR and phospho(y1248)-
ERBB2 in LNCaP cells. a Cells were harvested after 48 h of
experimental treatments. Lanes: (1) baseline, (2) paracrine entity, (3)
androgenic entity, (4) cells immunosegregated from coculture with
OHS cells, and (5) cells immunosegregated from coculture with
osteoblastic cells differentiated from human mesenchymal stem cells.
b Cells were treated with 50 ng/ml EGF or 10% FBS. Lanes: (1)
baseline, (2) EGF for 15 min, (3) EGF for 24 h, (4) EGF for 48 h, (5)
FBS for 48 h, and (6) 48 h untreated
490 Clin Exp Metastasis (2009) 26:485–496
123
We have previously evaluated the immunomagnetic cell
separation method for selective isolation of target cells and
demonstrated that target cell populations are highly enri-
ched [12–15]. But because kinase activity profiling might
be sensitive to the possible presence of contaminants,
we screened for phosphopeptide signatures generated by
immunoselected as well as monocultured OHS cells. Nota-
bly, the resulting substrate phosphorylation patterns were
closely similar for the two conditions (Table 3) and also
clearly distinguishable from those generated by the LNCaP
entities, which argues against significant contamination of
OHS cells in MOC-31-positive LNCaP cell isolates.
Fig. 3 Morphologic characteristics of LNCaP and osteoblastic cells.
a Monocultured LNCaP cells, untreated (magnification 9300). b
LNCaP/OHS coculture, at start of experimental incubation (magni-
fication 9400). The fluorescent cells are stained with FITC-
conjugated 9.2.27 antibody; accordingly, representing OHS cells. c
Monocultured OHS cells (magnification 9300). d Mononuclear cells
isolated from adult human bone marrow were cultured in mesencymal
stem cell-stimulating medium followed by osteogenic differentiation
medium for 20 days to obtain in vitro-differentiated osteoblasts
(magnification 9300). e The differentiated osteoblasts were fixed in
ice-cold methanol for 30 min, incubated in dark with 5% silver nitrate
solution for 30 min, and washed thoroughly with deionized water and
then with 5% Na
2
CO
3
and 0.2% formaldehyde, according to the von
Kossa staining protocol for calcium salts. Mineral deposits are
visualized as dark brown stain (magnification 9300). f Coculture of
LNCaP cells and in vitro-differentiated osteoblasts, at start of
experimental incubation (magnification 9400). The fluorescent cells
are stained with FITC-conjugated MOC-31 antibody; accordingly,
representing LNCaP cells. g Suspended cells binding MOC-31-coated
beads; accordingly, representing LNCaP cells, after immunomagnetic
selection from LNCaP/OHS coculture (magnification 9400) (Color
figure online)
Clin Exp Metastasis (2009) 26:485–496 491
123
Discussion
Given the considerable health care challenges generated by
the prevalence of metastatic bone disease in prostate can-
cer, the apparent shortage of adequate experimental models
to study this aspect of disease progression is striking. From
a clinical point of view, prostate cancer metastasis to bone
is a lengthy and complex disease process, which makes it
difficult to establish adequate experimental models to
recreate all steps involved. As highlighted in a compre-
hensive review [30], most of the experimental systems
examining this phenomenon are based on rodent models.
Importantly, the model systems used in this study exclu-
sively utilize cell types of human origin, but caution should
be exercised regarding any compelling conclusion based on
specific in vitro experimental conditions, which can not
reflect an intact microenvironmental in vivo setting. Nev-
ertheless, given that regulatory mechanisms implicated in
the metastatic phenotype are evoked when carcinoma cells
settle within an osteoblastic microenvironment, the com-
bination of our experimental models and analytical
technology may provide relevant information about func-
tional signalling networks that facilitate this biological
process, and in addition may enable the identification of
new targets for therapeutic intervention.
The OHS cell line was originally established from a
patient with aggressive osteosarcoma [4], and it might be
argued that it is not representative for physiological
osteoblasts. However, formation of osteosclerotic (i.e.,
osteoblastic) lesions following intratibial OHS cell inocu-
lation has previously been demonstrated by radiographic,
scintigraphic, and morphologic assessments [31].
Table 2 Peptide substrates with increased phosphorylation levels
generated by the LNCaP paracrine 1 entity and LNCaP cells immu-
nosegregated from coculture with OHS cells (co-LNCaP)
Peptide substrate Paracrine 1 Co-LNCaP
EGFR_y1197
a
1.50
b
2.16
EGFR_y1110 – 1.86
RB1_s807/s811 3.72 –
ERBB2_y877 1.06 –
ERBB2_y1248 – –
CDK2_t14/y15 1.47 –
EPHB1_y778 1.78 –
IRS2_y919 4.88 –
JAK1_y1022/y1023 1.81 –
LAT_y200 2.70 2.94
LCK_y394 1.38 –
MET_y1230/y1234/y1235 1.08 –
MST1R_y1353 2.29 2.20
MST1R_y1356/y1360 2.60 2.01
PDPK1_y9 1.50 –
PDPK1_y373/y376 1.50 –
PTK2_y576/y577 1.33 1.03
PTK2B_y579/y580 0.85 0.85
RASA1_y460 1.27 0.96
RET_y1029 1.24 –
ZAP70_y492/y493 1.68 1.21
CREB1_y134/s133 – –
ERBB4_y1284 – –
GSK3B_y216 – –
RAF1_s337/s338/y339/y340 – –
CHRNB1_y390 1.00 0.95
LTK_y772/y776/y777 – 0.96
DDR1_y792/y796/y797 1.11 0.91
MAPK10_t221/y223 1.28 0.98
MAPK12_t183/y185 – 2.23
PECAM1_y713 1.51 0.87
PRRX2_y214 1.23 0.93
ANXA1_y20/t23 1.93 –
CD79A_y182/y188 1.39 –
CTTN1_y477/y483 1.45 –
CTTN1_y499 1.64 –
ENO2_y43 1.43 –
EPHA2_y772 1.60 –
EPHA7_y608/y614 1.64 –
EPOR_y368 1.67 –
EPOR_y426 1.77 –
FER_y714 1.36 –
FES_y713 1.82 –
FGFR2_y769 1.28 –
FGFR3_y760 1.63 –
FRK_y387 1.47 –
LAT_y255 1.13 –
Table 2 continued
Peptide substrate Paracrine 1 Co-LNCaP
NTRK2_y702/y706/y707 1.08 –
PDGFRB_y579/y581 3.52 –
PDGFRB_y716 1.59 –
PIK3R1_y607/s608 1.59 –
PXN_y31 1.44 –
PXN_y118 1.61 –
TEC_y519 1.23 –
PFKFB1_s33 – –
PTPN11_y542 – –
SYN1_s9 – –
‘–’ denotes that the change in substrate phosphorylation level was not
found to be significant (P \ 0.05, Student’s t-test)
a
For each substrate, position of phosphorylation sites within the
protein is indicated
b
Fold changes (log
2
) relative to LNCaP baseline samples are listed
492 Clin Exp Metastasis (2009) 26:485–496
123
Kinase activity microarrays represent an emerging
technology that may become a powerful tool for signal
transduction profiling of biological samples. A key
advantage of this technology lies in its ability to provide
signalling pathway maps that indicate the state of infor-
mation flow through intracellular networks. Essentially, the
peptide substrate array technology determines kinase
activity profiles, which is different from but complemen-
tary to mass spectrometry, which identifies phosphorylated
proteins that represent the end products of kinase activity.
An additional advantage of kinase activity microarrays is
their robustness with small sample quantities, typically 2–
5 lg total protein being sufficient for Tyrosine Kinase
PamChip
Ò
Array analysis, in contrast to several milligrams
of total protein usually required for mass spectrometry
analysis.
Of note, the phosphopeptide signatures generated by the
LNCaP paracrine 1 and paracrine 2 entities and by OHS
cells cultured alone or with LNCaP cells, respectively, were
essentially identical. This indicates low variation between
biologically similar samples and a high degree of repro-
ducibility using this peptide substrate array technology.
Computational methods for mapping of phosphorylation
networks are being developed, and reconstruction of
EGFR-mediated pathways have been used to test applica-
bility of such models [32]. Correct network definition may
be restricted by the influence of contextual factors, such as
subcellular compartmentalization or temporal expression
of the proteins involved [33]. Therefore, to predict pathway
connectivity with sufficient accuracy, the use of more than
one computational technique on the dataset of interest has
been recommended [20]. In this study, two different
pathway visualization systems identified signalling net-
works that were essentially identical.
The signalling pathway mediated by EGFR was found to
be activated in androgen-sensitive LNCaP cells under the
influence of either osteoblastic cells or androgen treatment.
Table 3 Phosphopeptides generated by monocultured OHS cells
(mono-OHS) and OHS cells immunosegregated from coculture with
LNCaP cells (co-OHS)
Peptide substrates Mono-OHS Co-OHS
ANXA1_y20/t23
a
-1.56
b
-2.84
CD79A_y182/y188 -3.70 -4.54
CDK2_t14/y15 -0.94 -1.32
CHRNB1_y390 -0.49 -0.64
CREB1_y134/s133 -0.81 -1.29
CTTN1_y477/y483 -0.86 -1.43
CTTN1_y499 -1.56 -2.00
DCX_y112/s116 -0.92 -0.86
DDR1_y792/y796/y797 -0.40 -0.79
EFS_y253 -0.86 -1.18
ENO2_y43 -3.72 -4.76
EPHA2_y772 -1.43 -2.32
EPHA7_y608/y614 -1.60 -2.18
EPHB1_y778 -1.60 -2.40
EPOR_y368 -0.94 -1.40
EPOR_y426 -0.94 -2.32
ERBB2_y877 -1.74 -2.18
ERBB2_y1248 -1.22 -2.06
FER_y714 -1.15 -1.64
FES_y713 -1.03 -2.25
FGFR2_y769 -1.89 -
2.06
FGFR3_y760 -1.56 -0.89
FRK_y387 -1.22 -1.84
GSK3B_y216 -1.25 -0.97
JAK1_y1022/y1023 -2.18 -2.32
KRT6E_s59 -0.92 -1.47
LAT_y255 -1.29 -2.40
LCK_y394 -1.60 -2.25
LTK_y772/y776/y777 -0.86 -1.03
MAPK10_t221/y223 -0.94 -0.62
MET_y1230/y1234/y1235 -0.89 -1.12
NCF1_s315/s320 -0.67 -0.74
NTRK1_y496 -1.94 -2.84
NTRK2_y702/y706/y707 -2.94 -3.84
PDGFRB_y579/y581 -4.32 -3.84
PDGFRB_y716 -1.51 -1.00
PDGFRB_y771/y775/y778 -1.00 -1.25
PDPK1_y9 -1.40 -1.25
PDPK1_y373/y376 -1.36 -2.00
PECAM1_y713 -1.06 -1.47
PFKFB1_s33 -0.79 -0.92
PIK3R1_y607/s608 -1.25 -2.56
PLCG1_y771 -4.57 -4.64
PRRX2_y214 -1.36 -1.25
PTK2_y576/y577 -1.15 -0.81
PTK2B_y579/y580 -0.71 -1.51
PXN_y31 -1.25 -1.84
Table 3 continued
Peptide substrates Mono-OHS Co-OHS
PXN_y118 -1.03 -1.29
RAF1_s337/s338/y339/y340 -0.89 -1.18
RASA1_y460 -1.36 -1.09
RET_y1029 -1.22 -2.18
SYN1_s9 -1.06 -1.40
TEC_y519 -1.51 -1.64
TYRO3_y686 -1.40 -1.29
ZAP70_y492/y493 -1.56 -1.74
a
For each substrate, position of phosphorylation sites within the
protein is indicated
b
Fold changes (log
2
) relative to LNCaP baseline sample are listed
Clin Exp Metastasis (2009) 26:485–496 493
123
Analysis by conventional western immunoblotting also
revealed that ERBB2-dependent signalling was activated in
both the paracrine and androgenic LNCaP entities,
although the latter did not seem to generate this particular
substrate phosphorylation profile on the microarray analy-
sis. The kinase activity and protein expression data together
strongly indicates a central involvement of both signalling
pathways in the interaction between prostate carcinoma
cells and osteoblasts.
Additionally, given that EGFR and ERBB2 are phos-
phorylated in androgen-sensitive LNCaP cells upon
influence of osteoblasts, a functional androgen signalling
axis [16, 17] may appear to be permissive for activity of
these particular pathways in prostate cancer. Not the less,
EGFR phosphorylation resulting from short-term incuba-
tion with bone stromal conditioned medium (containing 6%
FBS) and suppression of in vivo bone metastasis formation
following targeted inhibition of EGFR-dependent signalling
have been demonstrated in androgen-independent prostate
carcinoma PC3 cells [34]. Separately, EGFR and ERBB2
may facilitate androgen receptor-driven activity in prostate
cancer at the level of target gene transcription in the absence
or at low concentrations of androgens [35, 36]. Yet,
androgen receptor pathway genes, identified by system-
level analysis of gene expression in primary tumor speci-
mens from therapy-naı
¨
ve prostate cancer patients, were
reported to be down-regulated, with a few exceptions, in
lymph-node metastases from the patients [37]. This finding
further supports the assumption that the regulatory control
by the androgen receptor on carcinoma cell biology is lost in
the process of prostate cancer metastasis, even in the pres-
ence of activating receptor ligands.
Of importance, our experimental data suggests that tar-
geted inhibition of the signalling pathways directed by
EGFR or ERBB2 may simultaneously ablate androgen-
driven proliferation of prostate carcinoma cells and the
survival responses within an osteoblastic microenviron-
ment. It equally provides a biological rationale for the use
of EGFR or ERBB2 inhibition in systemic prevention or
treatment of metastatic prostate cancer in the androgen-
sensitive stage of the disease. Intriguingly, the therapeutic
concept of EGFR or ERBB2 inhibition in hormone-
refractory prostate cancer has recently been evaluated;
however, in initial studies addressing the use of single-
agent therapies in patients with androgen-resistant disease,
neither receptor-blocking antibodies nor small-molecular
tyrosine kinase inhibitors showed clinically significant
activity [23–26].
Of note, the tumor suppressor PTEN phosphatase is
frequently found to be functionally inactivated in prostate
cancer, leading to increased activity of AKT kinase sig-
nalling independent of the up-stream EGFR/ERBB2 and to
insensitivity to EGFR/ERBB2 inhibitors [16, 38–40]. The
LNCaP cells are deficient in PTEN [41, 42], questioning the
relevance of extrapolating from our data that EGFR/ERBB2
signalling is involved in the maintenance of osteoblast-
induced survival responses in prostate cancer. On the other
hand, development of androgen independence and more
advanced disease is associated with increased PTEN loss
[43–45], favoring therapeutic EGFR/ERBB2 inhibition in
the initial, androgen-sensitive stage of the disease. Thus, if
exploitable in patients with therapy-naı
¨
ve prostate cancer,
inhibitory EGFR/ERBB2 targeting might be incorporated
into treatment schedules with a potential reduction in the
alternative requirement of long-term androgen depletion, a
reduction in related side effects, and, intriguingly, the
potential for an improvement in patient survival.
Acknowledgments We thank Dr. R. Reisfeld for the gift of the
425.3 and 9.2.27 antibodies. We are grateful to Ingrid J. Guldvik and
Heidi Rasmussen for technical assistance. This study was supported
by The Norwegian Cancer Society grant C-04083 and grants from
Faculty Division The Norwegian Radium Hospital (to A. H. Ree).
Bioinformatics service was provided by the Norwegian Microarray
Consortium at the national technology platform (http://microarray.no
), supported by the Functional Genomics Program of the Norwegian
Research Council.
Conflict of interest statements A. H. Ree, A
˚
. Bratland, P. J.
Boender, and R. Ruijtenbeek are owners of a patent for diagnostic
application of the Tyrosine Kinase PamChip
Ò
Array technology in
prostate cancer (WIPO no. WO/2008/125633). P. J. Boender and R.
Ruijtenbeek are employees of PamGene International BV.
Open Access This article is distributed under the terms of the
Creative Commons Attribution Noncommercial License which per-
mits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
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