Basic Mechanisms Responsible for Osteolytic and

Osteoblastic Bone Metastases

Theresa A. Guise, Khalid S. Mohammad, Gregory Clines, et al.

Clin Cancer Res 2006;12:6213s-6216s.

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Basic Mechanisms Responsible for Osteolytic and
Osteoblastic Bone Metastases
Theresa A. Guise,1 Khalid S. Mohammad,1 Gregory Clines,1 Elizabeth G. Stebbins,2
Darren H.Wong,2 Linda S. Higgins,2 Robert Vessella,3 Eva Corey,2
Susan Padalecki,1 Larry Suva,4 and John M. Chirgwin1

Abstract Certain solid tumors metastasize to bone and cause osteolysis and abnormal new bone formation.
The respective phenotypes of dysregulated bone destruction and bone formation represent two
ends of a spectrum, and most patients will have evidence of both. The mechanisms responsible
for tumor growth in bone are complex and involve tumor stimulation of the osteoclast and the
osteoblast as well as the response of the bone microenvironment. Furthermore, factors that
increase bone resorption, independent of tumor, such as sex steroid deficiency, may contribute
to this vicious cycle of tumor growth in bone. This article discusses mechanisms and therapeutic
implications of osteolytic and osteoblastic bone metastases.

Certain solid tumors, such as breast and prostate cancer, have a
propensity to metastasize to bone and cause osteolysis and
abnormal new bone formation (1, 2). The respective phenotypes
of dysregulated bone destruction and bone formation represent
two ends of a spectrum, and most patients will have evidence of
both. In fact, bone metastases are heterogeneous: data gleaned
from a rapid autopsy program indicate that the same prostate
cancer patient often has evidence of osteolytic and osteoblastic
disease as shown by histologic examination (3). The mecha-
nisms responsible for tumor growth in bone are complex and
involve tumor stimulation of the osteoclast and the osteoblast as
well as the response of the bone microenvironment. Further-
more, factors that increase bone resorption, independent of
tumor, such as sex steroid deficiency, may contribute to this
vicious cycle of tumor growth in bone, illustrated in Fig. 1. This
article discusses mechanisms and therapeutic implications of
osteolytic and osteoblastic bone metastases.
Breast Cancer: The Prototypic OsteolyticTumor
Breast cancer commonly metastasizes to and destroys bone,
causing pain and fracture. Tumors produce many factors that
Authors’ Affiliations: 1University of Virginia, Charlottesville, Virginia; 2Scios, Inc.,
Fremont, California; 3 University of Washington, Seattle, Washington; and
4
University of Arkansas for Medical Sciences, Little Rock, Arkansas
Received 4/24/06; revised 7/6/06; accepted 8/2/06.
Grant support: NIH grants R01CA69158, R01DK065837, and R01DK067333
(T.A. Guise), consortium grant from the Department of Defense to Emory
University, Prostate Cancer Foundation, Mellon Institute of the University of
Virginia, and Aurbach endowment of the University of Virginia.
Presented at the First Cambridge Conference on Advances in Treating Metastatic
Bone Cancer, a symposium held in Cambridge, Massachusetts, October 28-29, 2005.
Requests for reprints: Theresa A. Guise, Division of Endocrinology, Department
of Medicine, University of Virginia, Aurbach Medical Research Building, P. O. Box
801419, Charlottesville, VA 22903. Phone: 434-243-9284; E-mail: tag4n @
virginia.edu.
F 2006 American Association for Cancer Research.
doi:10.1158/1078-0432.CCR-06-1007
stimulate osteolysis: parathyroid hormone-related protein
(PTHrP), interleukin (IL)-11, IL-8, IL-6, and receptor activator
of nuclear factor-nB ligand (RANKL; refs. 4 – 9). Substantial data
support a role for bone-derived transforming growth factor-h
(TGF-h) and tumor-derived osteolytic factors, such as PTHrP, in
a vicious cycle of local bone destruction in osteolytic metastases.
Bone matrix stores several immobilized growth factors, partic-
ularly TGF-h, which is released in active form during osteoclastic
resorption (10) and stimulates PTHrP production by tumor
cells. PTHrP in turn mediates bone destruction by stimulating
osteoclasts. A dominant-negative mutant of the type II TGF-h
receptor inhibited TGF-h-induced PTHrP secretion in vitro and
development of bone metastases in an MDA-MB-231 experi-
mental metastasis model (5, 6). In addition, TGF-h regulates
several genes that are responsible for enhanced bone metastases
in MDA-MB-231: IL-11 and connective tissue growth factor (CTGF;
refs. 8, 9). Collectively, these studies provided proof of principle
to support a role for TGF-h blockade in the treatment of breast
cancer bone metastases.
SD-208, a small-molecule inhibitor of TGF-h receptor I
kinase, inhibits signaling downstream of the TGF-h receptor.
The in vivo efficacy of SD-208 was evaluated in an experimental
metastasis model using the breast carcinoma cell line MDA-MB-
231. SD-208 reduced the development and progression of
osteolytic bone metastases as assessed by computerized image
analysis of radiographs and prolonged survival. To dissect the
mechanism that regulates these in vivo therapeutic benefits, the
effects of SD-208 were analyzed in cultured MDA-MB-231 cells.
SD-208 did not affect cell proliferation. However, SD-208
inhibited TGF-h-induced smad2/3 phosphorylation as well as
decreased production of TGF-h-stimulated osteolytic factors
IL-11 and PTHrP and the growth-promoting factor CTGF. It also
reduced TGF-h secretion from MDA-MB-231 cells, suggesting
possible autocrine effects of TGF-h on the cancer cells. Taken
together, these data indicate that therapeutic targeting of TGF-h
may decrease the osteolytic bone metastases due to breast
cancer by blocking tumor production of osteolytic and growth-
promoting factors, such as IL-11, PTHrP, and CTGF.

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Fig. 1. Vicious cycle in metastatic bone cancer.Vicious cycle between prostate
cancer cells and bone. Once cancer cells arrive in bone, the four major players in
this vicious cycle include the cancer cells, osteoblasts, osteoclasts, and mineralized
bone matrix, a major source of immobilized growth factors. Prostate cancer cells
secrete factors, which stimulate osteoblasts to proliferate, differentiate, and secrete
growth factors. These factors are deposited into the bone matrix and also enrich the
local microenvironment of the tumor cells. Tumor cells secrete osteolytic factors,
most of which act via osteoblast production of the osteoclast differentiation factor
RANKL. Growth factors released from the mineralized bone matrix as a
consequence of osteoclastic bone resorption further enrich the local milieu. These
interactions reinforce each other to accelerate cancer progression.VEGF, vascular
endothelial growth factor; PDGF, platelet-derived growth factor.
Prostate Cancer: The Prototypic Osteoblastic
Tumor
Prostate cancer has a propensity to metastasize to bone and
locally disrupt normal bone remodeling. Although such
metastases have been classified as osteoblastic based on the
radiographic appearance of the lesion, it is clear that bone
resorption and bone formation are dysregulated. Recent
clinical evidence indicates that both processes contribute to
the metastatic phenotype even in the same patient. In fact,
high concentrations of the bone resorption marker N-
telopeptide were recently shown to predict a poor clinical
outcome in men with prostate cancer and bone metastases
(11, 12), and bone fractures predict poor survival in prostate
cancer patients (13). Thus, an understanding of the mecha-
nisms of tumor-induced bone formation and the role of the
osteoclast in this process is critical to improve therapy.
Mediators of osteoblastic disease are discussed herein as well
as preclinical data to target these mediators in combination
with antiresorptive therapy.
Osteoblastic bone metastases: molecular mechanisms of ET-1
action on osteoblasts. Tumor production of growth factors,
such as platelet-derived growth factor, insulin-like growth
factors, and adrenomedullin, has been implicated in osteoblas-
tic bone metastases (2, 14 – 20). Recently, several groups have
identified a role of the vasoactive peptide ET-1 in stimulating
the new bone formation associated with osteoblastic metastases
via the endothelin A receptor (ETAR) in mice and humans. An
ETAR antagonist (atrasentan) prevented osteoblastic bone
metastases in a mouse model and reduced skeletal morbidity
in men with advanced prostate cancer (15 – 18). However, the
molecular mechanisms through which ET-1 stimulates osteo-
blasts are unclear. Downstream targets of ET-1 in osteoblasts
were identified by gene array analysis of RNA isolated from
calvariae treated with or without ET-1 for 6 hours and 1, 4, and
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7 days using mouse Affymetrix (Santa Clara, CA) GeneChips
(22,600 genes). Up-regulated genes with possible roles in
osteoblast function included secreted factors (IL-6, Wnt5a,
TIMP-3, Cyr61, CTGF, and RANKL), signaling molecules
(SGK), and transcription factors (TSC-22, C/EBP d, TGIF, and
Twist 2). ET-1 significantly down-regulated Dkk1, a secreted
inhibitor of the Wnt signaling pathway recently implicated in
suppressed bone formation of multiple myeloma (21). Of these
factors, only IL-6, Cyr61, CTGF, TIMP-3, TGIF, RANKL, and
Dkk1 were validated in primary osteoblasts. Dkk1 abrogated
ET-1-induced osteoblast proliferation and new bone formation
in calvarial organ cultures but did not inhibit basal osteoblast
activity. Thus, ET-1 decreases Dkk1 expression and relieves
tonic-negative regulation of osteoblast activity (22). In multiple
myeloma bone disease, Dkk1 may depress bone formation.
The opposite may occur in osteoblastic disease, where ET-1
stimulates osteoblast activity by decreasing autocrine produc-
tion of the negative regulator Dkk1.
Osteoblastic bone metastases: role of PTHrP fragments. Pros-
ostate cancer metastases to the skeleton are frequently
osteoblastic despite abundant expression of the osteolytic
factor PTHrP. A proposed explanation for this paradox is that
NH2-terminal fragments of PTHrP stimulate new bone forma-
tion by activating the ETAR. PTHrP 1 to 16 stimulated
osteoblast proliferation and new bone formation in an ex vivo
calvarial organ culture assay. The response was equivalent to
that caused by equimolar ET-1, a 21-amino acid ligand for the
ETAR that potently stimulates new bone formation. ET-1
residues 6 to 9 (LMDK) and PTHrP residues 8 to 11 (LHDK)
share strong sequence homology that may explain the agonist
properties of both peptides (23). The effects on new bone area
and osteoblast number were blocked by ABT-627 (atrasentan),
an ETAR antagonist, which did not block new bone formation
stimulated by fibroblast growth factor-2. We found similarly
strong anabolic responses to PTHrP 1 to 20 and 1 to 23,
whereas PTHrP 1 to 34 instead caused extensive osteolysis.
These data show that NH2-terminal peptides of PTHrP exert
potent anabolic effects on bone via the ETAR. The NH2-terminal
peptides do not bind to ETA R when overexpressed in
mammalian cells, suggesting that an accessory receptor subunit
is expressed in certain cell types, such as osteoblasts, making
them responsive to these alternative ETAR ligands. The sequence
of PTHrP 18 to 23, RRRFF, is cleaved by prostate-specific
antigen. Proteolysis at this site frees residues 8 to 11 to bind to
ETAR, providing a molecular explanation for the osteoblastic
phenotype of PTHrP-positive prostate cancer bone metastases.
The mimicry of ET-1 by PTHrP NH2-terminal peptides suggests
that ETAR antagonists could be effective in treating prostate
cancer, both against the actions of endothelin itself and against
the anabolic effects of PTHrP fragments.
Osteoblastic bone metastases: role of osteoclastic bone resorp-
tion. Osteoclastic bone resorption also contributes to the
pathophysiology of osteoblastic metastases. Resorption
markers are increased (11, 12), and bisphosphonate anti-
resorptive drugs reduce skeletal morbidity in patients with
osteoblastic disease (24). To determine if osteoclast activity
contributes to skeletal morbidity in patients with prostate
cancer, we used a preclinical animal model of osteoblastic
prostate cancer to test the effect on tumor growth in bone of
osteoblast inhibition with atrasentan or osteoclast inhibition
with bisphosphonate (zoledronic acid) as single agents or in
6214s www.aacrjournals.org

combination. Intact male nude mice were inoculated with
LuCaP23.1, an androgen-responsive xenograft that secretes
prostate-specific antigen and ET-1. Mice were treated with
vehicle, atrasentan, zoledronic acid, or the combination
starting 1 week before tumor inoculation and continuing for
24 weeks. Mice treated with atrasentan or zoledronic acid
alone had less osteoblastic response compared with vehicle
but did not differ from each other. The combination of
atrasentan and zoledronic acid was more effective than vehicle
or single treatments with respect to end points of radiographic
imaging, histomorphometric assessment of tumor burden,
and serum prostate-specific antigen concentrations. The data
suggest that osteoblast and osteoclast activities cooperate to
drive the progression of prostate cancer growth in bone. Thus,
combination therapy targeting the two major bone cell types
should be an effective treatment for osteoblastic bone
metastases.
Role of the bone microenvironment: increased bone turnover
promotes tumor growth. The skeleton is the most common site
of metastases in patients with advanced prostate cancer.
Androgen ablation, a standard treatment for prostate cancer,
increases osteoclastic bone resorption and bone loss (25, 26).
To determine if this provides a more fertile environment for
bone metastasis, we developed two mouse models of metastasis
to bone that mimic the clinical scenario of men rendered
hypogonadal from treatment for prostate cancer. The first uses
PC-3 prostate cancer cells that result in osteolytic bone
metastases following intracardiac inoculation in nude mice.
The second model, TSU-Pr1, is a bladder cancer cell line that
results in mixed osteolytic-osteoblastic bone metastases. Hypo-
gonadal mice had bone loss due to increased osteoclastic bone
resorption and, when inoculated with the human prostate
cancer cell line PC-3 or the human bladder cancer cell line
TSU-Pr1, had accelerated bone metastases. Treatment with the
zoledronic acid prevented bone loss due to androgen depriva-
tion and also reduced metastases to bone. These data support
the hypothesis that increased bone resorption due to androgen
deprivation may result in a more fertile environment for the
development of bone metastases. Bone resorption inhibitors,
such as bisphosphonates, should prevent bone loss in
hypogonadal men with advanced prostate cancer and may also
decrease skeletal metastases.
Taken together, these data indicate that bone metastases are
the result of complex interactions among tumor cells, bone
cells, and the bone microenvironment. Targeting these inter-
actions should lead to effective treatment of this devastating
complication of advanced cancer.
Open Discussion
Dr. Pearse: You are getting massive bone formation in your
xenograft model. Do you think this new bone sequesters
growth factors, such as IGF and TGF-h, to be released upon
osteoclast resorption?
Dr. Guise: Wherever this bone formation occurs, there is
usually an osteolytic response as well. So when you form
abnormal, disorganized new bone, the body’s response is to
remodel that bone, and you get increased osteoblastic activity.
Dr. Vessella: You can see osteolytic and osteoblastic events
in the same section, but it is not concurrent. It is not that one
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Basic Mechanisms of Bone Metastases
lesion is osteolytic and the other lesion is osteoblastic. Within
the same lesion you can see both types of activities, so it is a
vicious cycle.
Dr. Roodman: What is the contribution of coupling in
addition to this? These are not totally uncoupled like myeloma.
Is there a contribution also simply to bone resorption followed
by bone formation? Is part of the role of bisphosphonates to
uncouple the process?
Dr. Guise: There is definitely a coupling response and the
bisphosphonates uncouple that. When you get blastic activity,
bone resorption follows. There may be additional contributions
of tumor osteolytic factors as well.
Dr. Lipton: What about PDGF and VEGF?
Dr. Guise: There’s some controversy regarding the role of
angiogenesis and bone metastases, because bone is already a
prevascular tissue; this is an understudied area but clearly an
important one. VEGF stimulates not only angiogenesis but
also osteoclastic bone resorption and osteoblast bone forma-
tion, so it could have multiple effects. Many of these angiogenic
factors, such as PDGF and VEGF, and also osteolytic factors,
such as IL-11 and IL-8, are regulated by HIF-1a, which is the
hypoxy-induced factor and like TGF-h may be an upstream
master switch to turn on many osteolytic factors. The angio-
genesis aspects of bone metastasis have been understudied in a
mechanistic way. We are creating a bone angiogenesis model to
look at some of the mechanisms by which osteolytic and
osteoblastic disease occur.
Dr. Clohisy: One of the things that has always bothered me
about the osteoblastic models is that they don’t look like
prostate metastases, they look more like osteosarcomas. Do you
think we have finished developing classic models? How would
you recommend we move forward?
Dr. Guise: What I showed you in terms of the prostate
models are about as good as we have right now, and they are
certainly not the best that we could have. We directly inject the
tumor cells into the bone, which is why it probably looks like
an osteosarcoma. We’ve tried to make it metastasize, like a real
metastasis model, but we have a ways to go.
Dr. Weilbaecher: In patients who have prostate cancer
bone metastasis, does it correlate that the higher the PSA the
worse the metastasis? Do PSA neutralizing antibodies exist
and could you use that in your models to confirm this
mechanism?
Dr. Guise: This is a difficult project, because it’s hard to
make the jump from in vitro to clinical significance. I don’t
know, but those of you who see cancer patients may know if
there is a correlation between PSA increase and the phenotype
of bone metastases.
Dr. Smith: I don’t know if that has been specifically
addressed. In men with hormone refractory prostate cancer
with bone metastases, markers of osteoclast and osteoblast
activity are highly correlated. It also appears that the most
well-differentiated tumors tend to make the most PSA and the
least well-differentiated tumors tend to form more lytic
metastases.
Dr. Weilbaecher: Do neutralizing antibodies exist?
Dr. Vessella: Neutralizing antibodies to PSA at this point in
time have not been able to affect what you see in bone. The PSA
that is produced exists in the serum as a complex, and only at the
local region is it an active enzyme. Apparently, we are not getting
enough of the antibody into the site to neutralize the active
6215s Clin Cancer Res 2006;12(20 Suppl) October 15, 2006
enzyme. It certainly drops the PSA level systemically, but it
doesn’t get into the bone site enough to neutralize the active PSA.
Dr. Roodman: We are finding more and more factors, but
do we have to target all of them to get rid of them or do we only
have to target a couple because they are all acting at subphar-
macologic levels?
Dr. Guise: It’s becoming so complex that just targeting one
factor or figuring out which is the most important is difficult
because all tumors are different. We need to understand what
factors are upstream of many of these things. Just targeting the
factors themselves is not going to be the whole answer. We are
going to have to use different therapeutic modalities to target
the cancer cells themselves.
Dr. Roodman: Why don’t we kill the osteoclasts and the
osteoblasts and not worry about all of the signal pathways?
Dr. Guise: I agree with you totally. We have to think about
this as we begin to understand or discover the complexity of the
system.

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