Molecular mechanisms of metastasis in prostate cancer

Abstract

Prostate cancer (PCa) preferentially metastasizes to the bone marrow stroma of the axial skeleton. This activity is the principal cause of PCa morbidity and mortality. The exact mechanism of PCa metastasis is currently unknown, although considerable progress has been made in determining the key players in this process. In this review, we present the current understanding of the molecular processes driving PCa metastasis to the bone.

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Asian Journal of Andrology
Molecular mechanisms of metastasis in prostate cancer
Noel W. Clarke, Claire A. Hart, Mick D. Brown
Genito-Urinary Cancer Research Group, School of Cancer and Imaging Sciences, Paterson Institute for Cancer Research,
Christie Hospital, University of Manchester, Manchester M20 4BX, UK
Correspondence to: Dr Mick D. Brown, Genito-Urinary Cancer
Research Group, School of Cancer and Imaging Sciences, Pa-
terson Institute for Cancer Research, University of Manchester,
Wilmslow Road, Manchester M20 4BX, UK.
Fax: +44-16-1446-3109 E-mail: mbrown@picr.man.ac.uk
Received: 10 October 2008 Accepted: 11 October 2008
Review
Abstract
Prostate cancer (PCa) preferentially metastasizes to the bone marrow stroma of the axial skeleton. This activity is the
principal cause of PCa morbidity and mortality. The exact mechanism of PCa metastasis is currently unknown, although
considerable progress has been made in determining the key players in this process. In this review, we present the current
understanding of the molecular processes driving PCa metastasis to the bone.
Asian Journal of Andrology advance online publication, 1 December 2008; doi: 10.1038/aja.2008.29
Keywords: bone, bone marrow stroma, metastasis, prostate cancer
1 Metastatic mechanisms in the primary tumour
The principal problem arising from prostate cancer
(PCa) is its propensity to metastasize. This tendency arises
from specic molecular mechanisms and interactions that
together lead to local invasion, extravasation and distal
migration from the primary site, followed by endothelial
attachment, transmigration and site-specic establishment
of metastases at secondary sites. Basic knowledge related
to this structured process has improved recently, but many
of the key elements are still poorly understood.
Local invasion is one of the fundamental early steps in
metastasis, as without it tumour spread cannot occur. To
develop invasive potential, the malignant cell must down-
regulate its cell–cell and cell–matrix adhesive characteristics,
become motile and acquire the ability to break down the
extracellular matrix (ECM) using degradative enzymes
[1]. Once the malignant cell has reached the interstitium,
it must enter the vascular or lymphatic circulation by
breaching the endothelial barriers. From there, the cell
must migrate via the blood or lymphatic circulation and
arrest at a secondary endothelial site before binding to the
endothelium, extravasating and transmigrating through
the endothelial layer to reach the interstitium, where it
proliferates and/or coalesces with other metastasized cells
to form a micro-metastasis (Figure 1) [2]. It will do this
only if the environment at the secondary site is favourable.
2 Primary site cell–cell adhesion
Maintenance of organic architecture depends on cell-cell
and cell-matrix binding. In the prostate and other structures,
a key cell–cell binding regulator is the cadherin–catenin
complex, whereas cell–matrix binding is largely mediated
by integrins, dimeric binding proteins comprising
a
-and
b
-chain subunits.
Cadherins are transmembrane glycoproteins, of which
E-cadherin is the best characterized in PCa. It serves
critical functions during embryogenesis and organogenesis
through intercellular adhesion and signaling [3]. The
locus coding for E-cadherin (16q22.1) is considered to
be a tumour-suppressor gene; loss of function enables
cell detachment and induces an invasive phenotype [4],
whereas transfection of E-cadherin complementary DNA
(cDNA) into invasive adenocarcinoma cells renders them
non-invasive [5, 6].
E-cadherin is attached intracellularly to the actin
cytoskeleton via intracellular catenin. Once anchored,
the transmembrane cadherins bind through their external
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domains to the binding sites of other cadherins on adjacent
cells. The cadherin–catenin complex is essential for both
morphogenesis [7] and subsequent structural and functional
organization of epithelia [8], and the disruption of either of
the interactive components produces signicant alterations
in cellular behaviour. E-cadherin has been extensively
studied in human cancers, resulting in its nomination as a
marker for metastatic biopotential in many tumours [9].
In primary PCa, reduced E-cadherin expression has been
correlated with increased tumour grade or stage, and with
bone metastasis and poor prognosis [10–12]. Further data
have confirmed the correlation with tumour grade, but
one study found no relationship between E-cadherin and
tumour progression or PCa death [13]. In animal models,
low E-cadherin expression has also been described in
both metastasizing and non-metastasizing PCa tumour
sublines [5]. An archival study of this issue [14] in paired
primary prostate tissue and prostatic bone metastases
from the same patients showed decreased expression of
E-cadherin messenger RNA (mRNA) in metastases in
nine of the total number of cases. The results suggest that
E-cadherin down-regulation, although important, is not the
foremost step in the metastatic cascade, but this protein
is a clinically relevant invasion–metastasis suppressor.
Indeed, it is a critical component in the general process of
epithelial to mesenchymal transition (EMT). For epithelial
cancer to progress and metastasize, cells must undergo
this transition, whereby cell polarity and cell–cell binding
are lost. These cells assume a mesenchymal phenotype, which
gives them the ability to invade the ECM and migrate to distant
Figure 1. Metastasis is characterized by proliferation, neovascularization and extravasation at the primary site. In the circulation,
malignant cells interact with the host immune system, typically resulting in cancer cell destruction or apoptosis. Surviving cells arrest
at secondary endothelial sites by a process of lectin binding consolidated by integrin-based stabilization of the epithelial–endothelial
binding. The cell then undergoes active transmigration. The binding process is complete within 30–60 min and transmigration within 24 h.
Once the cell reaches the interstitium, it may remain dormant for an undened period or it may coalesce with other cells and proliferate
to form a metastatic colony. This will then disturb local physiological function, leading to physiological dysfunction and anatomical
disruption. Any metastatic site may produce further metastases (Reproduced with permission from [2]).

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sites. This process involves the disruption of stable E-cadherin
binding, a primary event governing EMT, and it is accompanied
by increased expression of mesenchymal N-cadherin. The EMT
process may be an ‘on–off’ phenomenon, and it is possible that
transient functional down-regulation of E-cadherin may be a
feature of the metastatic process in PCa, with differences in
expression at the primary and secondary sites [14].
Integrity of the cadherin–catenin complex and its ancho-
rage to the actin cytoskeleton are required for E-cadherin-
mediated intercellular adhesion. Absence or dysfunction of the
catenin component of this complex may lead to impaired cellular
adhesion, despite apparently normal E-cadherin levels
[14, 15]. Clinical studies of PCa conrm the correlation
between catenin subtype expression with tumour de-
differentiation and local stage [16], although aberrant
expression of
a
-catenin is rare in the presence of normal
E-cadherin expression. A study of 28 prostatic tumours
found consistent abnormalities of E-cadherin and down-
regulation of
a
-catenin [17], and although Umbas et al.
[18] detected these effects in only four out of 52 cases, the
combination occurred in patients with advanced disease.
b
-Catenin has dual functions in prostatic and other
tissues. In addition to its role in the cadherin–catenin com-
plex, it also regulates signal transduction by binding to DNA
and activating gene transcription. Few reports describe
abnormal
b
-catenin signalling as a master regulator of PCa
(< 4% of primary prostate tumours have
b
-catenin mutations
[19]), but aberrant
b
-catenin expression seems to affect the
function of the cadherin–catenin complex. This notion is
supported in a paired primary or bone metastasis study
[14], in which 13 out of 14 primary tumours had high
b
-catenin expression, whereas 12 out of 14 metastases
showed down-regulated
b
-catenin mRNA levels compared
with their primary tumours. Therefore, there is a striking
contrast in the levels of
b
-catenin mRNA between primary
tumours and metastases, suggesting a major dysfunction
of the cadherin–catenin complex. This factor may be an
important early step in the metastatic process. There are,
however, unexplained observations that run counter to
this hypothesis, for example,
b
-catenin expression in the
primary tumour does not appear to reect the metastatic
potential of tumours in some patients, and E-cadherin
is not lost from metastatic cells, although it may be re-
expressed in the secondary site once it is lost in the pri-
mary site. Better understanding of this process is required,
but, overall, these observations suggest that the essential
E-cadherin–
b
-catenin complex is often impaired during
metastasis.
3 Cell–matrix adhesion and matrix degradation
Integrins are essential for cell–matrix attachment.
Integrin expression varies between tumours, but over-
expression of
a
6
and
b
3
integrins have been associated
with increased invasion [20, 21]. This may suggest the
anchorage of the malignant cell to the basement membrane
(BM) or the involvement of signalling pathways related
to cell motility. Whatever the actual mechanism, integrins
are fundamentally important in the binding and migration
processes at metastatic sites, where they work together with
enzymes that degrade the ECM and BM. These structures
are composed mainly of type IV collagen, laminin, b ro nec-
tin, entactin and tenascin [22]. Leucocytes and malignant
cells are thought to be the only cells that are able to breach
the BM, a process facilitated by the production of matrix
metalloproteinases (MMPs). Twenty-four MMPs have been
described to date [23] and they act to degrade the ECM. MMP
activity is regulated by tissue inhibitors of metalloproteinases
(TIMPs), and imbalances in the MMP:TIMP ratio due to
either TIMP down-regulation or increased MMP production
by tumour cells can induce an invasive phenotype [24]. In
metastasis, this balance is vitally important both in endothelial
barrier degradation [25] and in the establishment of metas-
tases wi thin bone marrow stroma (BMS) [26, 27] (Figure
2). The proteolytic enzyme, urokinase-type plasminogen
activator, is also important in the MMP cascade. It has direct
lytic activity on bronectin, and through plasmin it activates
procollagenases. It works to initiate MMP action and is
particularly critical in the development of PCa metastases [27].
4 Cell migration and motility: the GTPase axis
Cell motility and migration in prostate and other
cancers are linked integrally to Ras and other GTP-binding
proteins, for example, Rho and Rac. These proteins
are important for general cellular functions, including
cytoskeletal assembly, intracellular signalling and physical
movement of cell membranes and whole cells [28]. Ras
is a transmembrane glycosylated protein that regulates
downstream cellular activities such as cell proliferation,
nuclear transcription, apoptosis and invasion [29] (Figure 3).
It acts as a membrane transducer, as extracellular signals
bind to receptor tyrosine kinases, which in turn activate
Ras and initiate downstream events [30, 31]. The Ras
family, which has a major influence on cell signalling,
comprises h-ras, k-ras, n-ras, r-ras and m-ras, and
although Ras mutations are rare in PCa (3%), they are
associated with 30% of solid tumours [31].
The Rho GTPases are similar to Ras in their structure
and synthesis; their activation lies downstream of Ras
and is therefore Ras-dependent. This family currently
comprises RhoA, B, C, E and G; Rac1, Rac2, cdc42-H5
and TC10, all proteins involved in cell motility. It has
been suggested [32] that Rho GTPases act through actin
dynamics, guiding morphological changes, including
cell growth and movement. Cell movement may occur

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via a ‘molecular clutch’, which involves the extension
of filopodia bound to the cortical actin network and a
fixed extracellular ligand, resulting in net movement of
the whole cell (Figure 4). Prevention of Rho synthesis
or activity should result in reduced cell motility, with a
corresponding reduction in invasion across endothelial
Figure 3. Schematic diagram showing the Ras signalling pathway and its linkage to the GTPase cellular motility axis (Reproduced with
permission from [2]).
Figure 2. (A): Photomicrograph showing matrix metallo proteinase (MMP)-7 staining of prostate cancer (PCa) cells in culture. High
MMP-7 staining is seen at the leading edge of the cell relative to the rufing border at the margin of the pseudopodial extension (arrows);
original magnication (× 400), scale bar = 10
m
m. (B): Confocal 3D imaging of PCa cells in bone marrow co-culture showing PCa
invasion of bone narrow stroma (BMS). False colour image of a PC-3 cell within the BMS: Blue being 0
m
m, closest to the viewer (top
of BMS) through to red 10
m
m, furthest away from the viewer (bottom of the BMS layer). Using morphology for identication, the
arrow (i) shows the leading pseudopodia of a PC-3 cell, with a second arrow (ii) showing the trailing end. (C): 3D image of the same
picture showing the PC-3 cell underneath the BMS. MMP concentrations are highest at the leading edge of the cell. The cells also have
the extended mesenchymal morphology typical of motile cells. Scale bar = 10
m
m (Reproduced with permission from [2]).
barriers. This Ras–Rho-mediated activity is thought
to be important in cellular migration and metastasis in
prostate and other cancers. A study using bisphosphonates
to inhibit the mevalonate pathway (and thus RhoA) in
PCa [25] showed that cell motility and transmigration of
PCa cells across human bone marrow endothelial (BME)

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barriers and human BMS were inhibited in the presence of
zoledronic acid. A further study that examined the effects
of inhibiting the farnesyl and geranyl–geranyl prenylation
pathways [33] showed that migration and motility of
PCa cells were reduced dramatically by inhibition of Ras
prenylation (and therefore also inhibition of Rho acti-
vation). It is likely that the Ras–Rho axis is activated in
PCa metastasis and that this underpins the acquisition of
cell motility that is fundamental for successful metastasis.
5 Prostate tumour cell clearance from peripheral
blood
In solid tumours, malignant cells enter the circulation
increasingly as the tumour load grows. Iatrogenic cel-
lular shedding into the circulation occurs in clinical
situations, for example, during transurethral resection
of the prostate (TURP) [34, 35], radical prostatectomy
[36], prostate biopsy [37] and brachytherapy [38]. This
cellular dissemination is unexpectedly not associated with
a perceptible increase in metastasis development, perhaps
because of the inability of individual cells to propagate or
because of other unknown factors. Whatever the reality,
tumour growth is accompanied by an ongoing process
of cellular clearance from the circulation. Some authors
suggest that this cell clearance takes up to 4 weeks [37],
but this proposed time scale is far too long and the speed
of cell clearance from the circulation is almost certainly
more rapid. Chambers et al. [26] proposed that clearance
is mainly attributable to the arrest of relatively large
epithelial cells (or cellular clumps) in the first capillary
bed they encounter. However, this cannot be the sole
explanation; if it were, the incidence of pulmonary and
hepatic metastases would be much higher than it actually
is in PCa and other cancers. There must be additional
relevant factors, predicated on the differential binding
of PCa cells and differences in chemo-attraction, that
activate cellular motility. In vitro models of prostate
epithelial cell (PEC) binding to the human BME have
shown that the process of epithelial–endothelial binding is
virtually complete within an hour. Once this has occurred,
epithelial cell migration through the endothelial barrier
occurs within 24 h [39] (Figure 5). These laboratory
ndings are supported by reverse transcriptase–polymerase
chain reaction-based measurements in men undergoing
TURP, showing that PECs appear in the circulation upon
commencement of surgery, but are undetectable within 2
h of the procedure’s conclusion [40]. It is clear, therefore,
that once a cell enters the circulation, it is rapidly taken out,
probably by endothelial surface binding at a secondary site.
6 Distal attachment and tumour cell transmigration
through the endothelium
Tumour cells arrest on endothelial surfaces within the
circulatory system and subsequently undergo transen dothelial
Figure 4. (A): Optical image of a motile prostate cancer (PCa) cell
with a lamellipodial extension (L) projecting out in the direction of
cellular travel. N, Nucleus; D, de-polymerized actin laments. (B):
Schematic showing the internal architecture of the lamellipodium
(L), with actin laments linking the internal cellular structure to
the external lipid cellular membrane. These laments undergo a
constant process of polymerization and destruction (D), resulting
in movement of the lamellipodium and forward movement of the
cell (Reproduced with permission from [2]).
Figure 5. (A): Photomicrograph of a bone marrow endothelial
(BME) monolayer (phase contrast) seeded with prostate cancer
(PCa) PC-3 cells transfected with green uorescent protein (GFP)
(green). Cells bind to the junctional endothelial areas within 30–60
min. Thereafter they induce BME retraction and migrate into the
interstitium. Scale bar = 10
m
m. (B): This process involves active
cellular movement and cellular expansion as shown by the time-
lapse volumetric reconstructions of GFP-marked cells as they
extravasate through the endothelial monolayer (Reproduced with
permission from [2]).

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migration; this is a key event in cancer metastasis. Tumour
cell–endothelial interactions involve multiple adhesive
interactions (docking and locking) at the molecular level
[21]. The initial step is thought to involve selectins, fol-
lowed by stabilization through integrin binding [41].
These are not the only binding steps, because antibodies
to CD11a, CD18, LFA-1 and CD31 have been shown to
interfere with the binding process [42].
Site-specific adhesion determinants play a role in
preferential metastasis to individual organs. Molecules
postulated to be involved in tumour–endothelial adhesion
include platelet–endothelial cell adhesion molecule-1
(PECAM-1 or CD31) [43],
a
4
b
1
integrin and sialyl Lewis
X, which bind to the endothelial cells through E-selectin,
vascular cell adhesion molecule 1 (VCAM-1) [44] and
others.
Tumour cells penetrate endothelial junctions after
adhering to the surface of endothelial cells (Figures 5 and
6). Endothelial cells appear to be actively involved in trans-
mi gration, as dynamic changes occur in the expression and
localization of adhesion molecules, including N-cadherin,
VE-cadherin and PECAM-1 [45], inducing endothelial
cell retraction once the tumour cell adheres to the under-
ly ing ECM. Binding to laminin, type IV and type V
collagens is mediated by
b
1
and
b
4
integrins, whereas
bin ding to hyaluronan, fibronectin, type I collagen and
cellular migration is mediated by
b
1
integrins and CD44
[44, 46–49]. Understanding the process of secondary site
binding and endothelial transmigration in PCa has been
facilitated by the development of co-culture models using
human BMS and primary PECs and PEC lines [27, 39]
and the establishment of prostate epithelial colonies in
human BMS [50, 51]. These models have demonstrated
that cell–matrix binding depends fundamentally on the
b
1
integrin component of the integrin-binding mechanism.
Studies using various endothelial types as well as benign
and malignant PECs have shown that PECs bind more
avidly to bone marrow endothelial cells (BMECs) than to
other endothelia and that benign and malignant cells have
the same binding capacity for those endothelial surfaces.
Why, then, do metastases not develop from the PECs
known to be present in the circulation during prostatic
resection for benign prostatic hyperplasia (BPH)? The
answer to this question lies in the differential ability of
PECs to migrate across the endothelial barrier. In vitro
studies using green uorescent protein (GFP)-transfected
PCa cells in conjunction with time-lapse confocal micro-
scopy have enabled cellular tracking measurements of
benign and malignant PECs in epithelial–endothelial co-
culture [39] (Figures 5 and 6) and have shown that only
malignant cells will transmigrate through the endothelial
layer. Benign cells will bind in the same way as malignant
cells, but they do not cross the endothelial barrier into the
interstitium [39].
Explication of this mechanism is critical to the un-
der stand ing and potential treatment of metastases in
PCa. Once bound, PECs induce rapid endothelial cell
retraction (Figure 6), but the precise mechanism inducing
this reac tion is at present unclear. A major component of the
sig nal ling cascade modulating endothelial permeability is
intracellular Ca
2+
[52]. Studies by Lewalle et al. [53] showed
that binding of breast epithelial cells to human umbilical vein
endothelium cells (HUVECs) induced a transitory rise in
the HUVEC intracellular Ca
2+
concentration, resulting in
endothelial retraction and epithelial migration. This Ca
2+
rise and the process of endothelial retraction are entirely
dependent on cell–cell contact, and inhibition of the Ca
2+
elevation inhibited breast epithelial cell transendothelial
migration. Binding of PECs and melanoma cells also
induces increases in intracellular Ca
2+
levels [54], correlat-
ing with increased binding of the epithelial cells. Further
studies of calcium-binding agents support this notion:
Figure 6. Reconstructed confocal microscopic image of prostate cancer (PCa) cells transfected with green uorescent protein (GFP)
(green) seeded onto a bone marrow endothelial (BME) monolayer (grey) and photographed sequentially with time-lapse confocal
microscopy. (A): PCa cells binding to the junctional areas of the endothelium. (B): The endothelial cells have started to retract,
leaving gaps in the endothelial barrier. (C): The epithelial cells then migrate through into the underlying interstitium (Reproduced with
permission from [2])

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treatment of BMEC lines with zoledronic acid, a potent
calcium-chelating agent, tightens endothelial–endothelial
cell binding and limits transmigration [25].
The effect of Ras–Rho inhibition of reducing the propen-
sity of PECs to invade across endothelial barriers suggests that
a major component affecting cancer cell migration is inhibition
of transduction pathways related to the Rho axis. This
inhibitory effect has been demonstrated in vitro in PCa using
zoledronic acid [25] and the prenylation inhibitor AZD3409
[33, 55]. The inhibitory effects of these compounds are
known to be related to Rho through its interaction with
Ras [56]. Inhibition of this pathway affects downstream
prenylation of small GTPases (Rho–Rac), which are
known to have an integral involvement in cell motility.
Therefore, an early event following integrin
b
1
binding in
PCa cells may be the induction of specic pathways that
relate to Ras and subsequently Rho–Rac. These in turn are
associated with the epithelial-to-mesenchymal transition,
a phenomenon known to be important in the cellular
migration process.
7 Chemo-attraction at the secondary site: chemokines
and lipids
The ‘seed–soil’ hypothesis of Paget is exemplied by
PCa, with its predilection for the red bone marrow of the
axial skeleton [57]. Once there, the malignant cells disturb
integrated and balanced skeletal functions and displace the
red bone marrow, inducing marrow dysfunction and bone
marrow failure. Two factors contribute to this homing
phenomenon: the presence of chemokines and the afnity
for energy-rich sources, such as specific lipids that are
freely available within red marrow adipocytes.
The chemokine axis is important in the homing of
haematological and immunological cells to specic targets.
Cells from various epithelial tumours share many of the
trafcking characteristics of this haematopoietic stem cell
(HSC) homing system [58]. Homing of the HSCs to the
bone marrow during foetal life and after bone marrow
transplantation has been well characterized. The key
molecular axis for this process was identied as the CXC
chemokine stromal-derived factor-1 (SDF-1 or CXCL12)
and its receptor CXCR4 (CD186). This model is supported
by the knowledge that both BMECs and osteoblasts ex-
press SDF-1 [59–61], the observation that CXCR4 knock-
outs do not show haematopoietic engraftment of the
bone marrow [62] and the recognition that the level of
CXCR4 expression in HSCs determines their ability to
engraft the bone marrow [63]. The CXCR4–SDF-1 axis
is also known to play an important role in targeting solid
tumour metastases to the bone marrow. This is important
in various primary tumours, including the breast [58],
kidney [64], lung [65], pancreas [66] and prostate [57, 67].
In vitro, CXCR4 and SDF-1 are involved in the motility
process: interactions alongside CCR7 or CCL21 trigger
pseudopodial invasion by malignant breast epithelial cells
through actin polymerization [58]. These results have
led to the hypothesis that CXCR4 is the key component
of metastatic implantation in the bone marrow and that it
represents an important therapeutic target for metastatic
bone disease in PCa and other cancers. Indeed, blockage
of CXCR4 signalling in breast cancer by neutralizing
antibodies [58] or peptide antagonists such as T140 [68]
has been shown to inhibit metastasis in vivo. Sun et al.
[67] also showed that CXCR4 expression increased with
increasing prostatic malignancy; the greatest expression
was observed in aggressively metastatic PC-3 cells and
in human bone metastasis specimens. This gradient of
expression suggests that CXCR4–SDF-1 signalling may be
a key signalling pathway for metastatic spread to the bone.
It has also been demonstrated [57], using a matrigel BM
invasion assay, that SDF-1 signalling induced both DU145
and PC-3 cells to invade. However, Hart et al. [39] used
recombinant SDF-1 and T140 inhibitors to show that the
CXCR4–SDF-1 signalling pathway is not the sole chemo-
attractant important for the spread of PECs to the bone,
conrming that, although SDF-1 is a potent stimulus for
invasion, the level of invasion it induces is signicantly
less than that seen using either BMECs or BMS alone.
This phenomenon was reinforced in these experiments by
the observation that the use of a specic CXCR4 antagonist
peptide (T140), at a concentration that blocked PEC invasion
in response to maximum levels of SDF-1 signalling, did not
completely block invasion towards either BME or BMS. Thus,
although the CXCR4/SDF-1 signalling pathway is important
in PCa metastasis, it is not the only chemokine signalling
pathway involved [39].
Another important stimulus is the requirement for the
metastasizing PCa cells to seek a lipid source. Cancer cells
are in a state of rapid metabolism and have a fundamental
requirement for lipids, to be used either as an energy source
or in the processes involved in tumour cell maintenance,
proliferation and migration. An in vitro study [69] showed
that PC-3 cells grew rapidly in the vicinity of lipid cells
in bone marrow, and further studies [70] showed that PCa
cells take up lipids rapidly as soon as they are seeded onto
the human bone marrow (Figure 7). Specific lipids also
act as strong chemo-attractants for PCa cells. Treatment
with arachidonic acid, an omega-6 lipid, results in rapid
migration of PC-3 cells towards bone marrow stroma, an
effect that is blocked competitively using omega-3 lipids.
Further experiments of lipid depletion in the bone marrow
conrm this effect: the attractiveness of the human bone
marrow to PCa cells decreased dramatically once the BMS
was depleted of lipid cells prior to epithelial seeding [70],
confirming that specific lipids are critical to metastasis

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and that they may be an important determinant of the site
specicity of the bone marrow in PCa.
8 Molecular mechanisms of metastatic prostate
cancer in the bone or bone marrow
Once established at the secondary site, prostatic
micro-metastases develop in the bone marrow space,
often in close association with the bone surface, where
the osteoblast–fibroblast microenvironment is disturbed
locally. It is postulated that the rst event in this meta-
static developmental process is osteoclast-mediated
bone resorption, leading to the release of stimulatory
cytokines from the bone surface and inducing a cycle of
resorption or tumour stimulation, but this hypothesis has
not been proven denitively. As the metastasis develops,
an imbalance occurs in the regulated, coupled skeletal
cycle of bone resorption and bone formation, resulting
in accelerated and synchronous bone formation and
resorption. This is caused by changes in local cytokine
production and interactions (Figure 8).
Many stimulating factors have been identified with
respect to osteoblastic metastases in PCa. These mecha-
nisms have been clarified in recent years and point
towards the importance of the endothelin axis. There
are three types of endothelin (ET-1, -2 and -3), which act
through the endothelin receptors ETa and ETb. They are
synthesized in vascular endothelial cells and are involved
in processes such as vasoconstriction, nociception and
the physiological regulation of bone function, amongst
others. Effects on bone function are important in relation
to PCa. Nelson and Carducci (reviewed by Nelson [71])
showed that exogenous ET-1 induces PCa proliferation
and enhances the mitogenic effects of insulin-like growth
factor (IGF) and epidermal growth factor. Regarding PCa
metastasis in bone, ET-1 production is a major factor in
osteoblast overstimulation [72]. PECs produce ET-1, and
its receptor, ETa, is present throughout the prostate gland
[73–75]. ET-1 is also produced by PCa cells in a bone
environment [76]. Experiments using an osteoblast mouse
model [72] showed that tumours producing ET-1 (e.g.,
PCa) act via ETa receptors on osteoblasts to stimulate
accelerated bone formation. This abnormal activity is
blocked by the ET-1 inhibitor ABT-627 (Atrasentan) [71].
Although ET-1 is important, it is not the only osteoblast
stimulator in PCa metastasis. Other factors include up-
regulation of the Wnt pathway and production of cytokines,
for example, bone morphogenetic protein, TGF-
b
, IGF,
Figure 7. (A): Photomicrograph showing lipid uptake by a prostate cancer (PCa) cell in co-culture. (B): The lipid droplets (Oil Red
O-stained vesicles) are taken up rapidly by the PCa cell and have been shown to be intracellular using confocal microscopy. (C): At
higher magnication, the lipid droplets are more clearly demonstrated to be inside the PCa cells after co-culture with human BMS.
The PCa cells have an afnity for the adipocytes and co-localize with them in the bone marrow. Scale bars = 10
m
m (Reproduced with
permission from [2]).
Figure 8. Photomicrographs of two bone biopsies taken from
different prostate cancer (PCa) patients. These show the typical
appearance of early micro-metastases from PCa. The epithelial
cells are stained for prostate-specic angiten (PSA) and appear
brown in this image. The cell colonies lie close to the bone
surface (uniformly pale), where they stimulate early osteoblastic
activity. In the surrounding bone marrow, the fibroblasts are
stimulated to induce a desmoplastic reaction. There is no evi-
dence of bone resorption at this stage of the disease process
(Reproduced with permission from [2]).

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vascular endothelial growth factor, platelet-derived growth
factor and MDA-BF [77]. A further interesting aspect of the
cytokine balance in PCa metastasis relates to the IGF axis
and parathyroid hor mone-related protein (PTHrP), which is
produced in PCa bone metastases [78]. The prostate-specic
antigen (PSA), a known protease, cleaves PTHrP and possibly
shifts the balance within the immediate milieu of the prostate
metastasis from bone resorption to formation [79, 80]. PSA
can also cleave insulin-like growth factor binding protein
(IGFBP-3), which in turn increases the levels of IGF-1. This
too would have the effect of shifting the axis of stimulation
by the metastatic PCa cells towards increased osteoblast
activity [81]. Osteoblast hyperactivity is responsible for the
measurable increase in bone volume in PCa bone metastases
[82, 83] and for the accelerated bone mineralization rate [84].
Prostate tumour-generated bone in these deposits is formed
as abnormal ‘woven’ bone, characteristic of the bone
produced in high-turnover states. This is responsible for
the sclerotic appearance measured histomorphometrically
[83] and seen radiologically in over 90% of patients with
advanced metastatic PCa [85].
The traditional view of PCa as osteoblastic obscured
for many years the fact that the disease is responsible for
major bone destruction. Resorptive effects of PCa were
initially suggested following histological studies in bone
[86], and the phenomenon was subsequently confirmed
after histomorphometric measurements of metastatic bone
biopsies [82, 83] and biochemical measurements of bone
resorption products in humans [87, 88]. The paradox of
increasing bone volume in the presence of bone resorption
is explained by histomorphometric studies showing that
the resorption of the existing skeleton is accompanied by
synchronous replacement of abnormal woven bone, which
itself undergoes further resorption [83]. This produces
a measurable increase in bone volume coincident with
wholesale destruction of the normal skeleton.
Molecular mechanisms responsible for this lytic
process arise as the consequences of abnormal concen-
trations of soluble growth factors produced by the
invad ing PCa, which stimulate abnormal osteoclast
ac ti vit y, in du cin g bo n e re s or pt i on . O st eo c la st
recruitment, differentiation and activation by tumours are
incompletely understood, but are known to be related to
the osteoblast stimulation that results from osteoblastic
over-expression of NF-κB (RANK ligand) and the
production of osteo protegerin, known to be increased in
PCa metastasis [89]. This effect may also be induced
by macrophage colony-stimulating factor, the receptor
activator of the RANK ligand and osteoprotegerin [90,
91]. Osteoblasts secrete the RANK ligand, which then
induces osteoclast differentiation by binding to the
RANK surface receptor on the osteoclast precursor, which
in turn stimulates osteoclastogenesis [90]. Osteoprotegerin
plays a key regulatory role in this process by competing
for the RANK-binding site on osteoclast precursors. A co-
factor in this process is PTHrP. Cancer cells are unable to
express the RANK ligand and therefore cannot stimulate
osteoclastogenesis by this route. However, when PTHrP
is present (as in murine osteoblasts and haemopoietic
progenitors in culture [92]), osteoclasts differentiate in
the absence of other stimulatory agents, suggesting that
PTHrP plays a facilitating role. PTHrP is a major factor in
bone resorption in breast cancer [93] and is expressed in
both primary tumours and bone metastases of PCa [78].
9 Conclusion
The molecular mechanisms of metastasis in PCa
are complex and involve a number of specic steps and
interrelated mechanisms. A more complete understanding
of the molecular mechanisms controlling this process
will help to develop novel therapies that may enable us to
control this progressively fatal condition.
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