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review article

mechanisms of disease

Mechanisms of Bone Metastasis

G. David Roodman, M.D., Ph.D.

one metastases are a frequent complication of cancer, occurring in up to 70 percent of patients with advanced breast or prostate cancer1
and in approximately 15 to 30 percent of patients with carcinoma of the lung,
colon, stomach, bladder, uterus, rectum, thyroid, or kidney. The exact incidence of bone
metastasis is unknown, but it is estimated that 350,000 people die with bone metastases
annually in the United States.2 Furthermore, once tumors metastasize to bone, they are
usually incurable: only 20 percent of patients with breast cancer are still alive five years
after the discovery of bone metastasis.3 The consequences of bone metastasis are often
devastating. Osteolytic metastases can cause severe pain, pathologic fractures, lifethreatening hypercalcemia, spinal cord compression, and other nerve-compression syndromes. Patients with osteoblastic metastases have bone pain and pathologic fractures
because of the poor quality of bone produced by the osteoblasts. For all these reasons,
bone metastasis is a serious and costly complication of cancer.

From the Department of Medicine, Division of HematologyOncology, University

of Pittsburgh, the University of Pittsburgh
Cancer Institute, and the Department of
Veterans Affairs Medical Center all in
Pittsburgh. Address reprint requests to Dr.
Roodman at the University of Pittsburgh,
School of Medicine/Hematology, Kaufmann
Medical Bldg., Suite 601, 3471 5th Ave.,
Pittsburgh, PA 15213, or at roodmangd@
msx.upmc. edu.
N Engl J Med 2004;350:1655-64.
Copyright 2004 Massachusetts Medical Society.

types of bone metastasis

Metastases have been characterized as osteolytic or osteoblastic (Fig. 1). This classification actually represents two extremes of a continuum in which dysregulation of the
normal bone remodeling process occurs (Fig. 2). Patients can have both osteolytic and
osteoblastic metastasis or mixed lesions containing both elements. Most patients with
breast cancer have predominantly osteolytic lesions, although at least 15 to 20 percent
of them have predominantly osteoblastic lesions.4 In addition, secondary formation of
bone occurs in response to bone destruction. This reactive process makes it possible to
detect osteolytic lesions by means of bone scanning, which identifies sites of active
bone formation. Only in multiple myeloma do purely lytic bone lesions develop. In contrast, the lesions in prostate cancer are predominantly osteoblastic.5 However, there is
also increased bone resorption in the osteoblastic lesions of prostate cancer, and agents
that block bone resorption can decrease bone pain and the risk of pathologic fractures.6

bone as a preferred site of metastasis

Several factors account for the frequency of bone metastasis. Blood flow is high in areas
of red marrow,7 accounting for the predilection of metastases for those sites. Furthermore, tumor cells produce adhesive molecules that bind them to marrow stromal cells
and bone matrix. These adhesive interactions cause the tumor cells to increase the production of angiogenic factors and bone-resorbing factors that further enhance tumor
growth in bone.8 Bone is also a large repository for immobilized growth factors, including transforming growth factor b, insulin-like growth factors I and II, fibroblast growth
factors, platelet-derived growth factors, bone morphogenetic proteins, and calcium.9
These growth factors, which are released and activated during bone resorption,10 pro-

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vide fertile ground in which tumor cells can grow.

This seed-and-soil hypothesis of the mechanism
of bone metastasis was first advanced by Stephan
Paget in 188911 and is supported by findings in animal models of bone metastasis.

A
Osteoclast

control of normal
bone remodeling
Osteoblasts

The adult skeleton continually turns over and remodels itself through the coordinated activity of osteoclasts and osteoblasts on trabecular surfaces and
the haversian system. In normal bone, there is a balanced remodeling sequence: first, osteoclasts resorb bone, and then osteoblasts form bone at the
same site.

osteoclasts

Figure 1. Osteoclasts and Osteoblasts in Normal Bone

and Bone Metastasis.
Panel A shows osteoclasts and osteoblasts in normal
bone (toluidine blue, 100). The large osteoclast is actively resorbing bone. Osteoblasts are small, cuboid cells
that actively lay down bone matrix. Panel B shows osteolytic bone metastasis (hematoxylin and eosin, 200). Renal carcinoma cells are invading the bone marrow, and
osteoclasts (arrows) are actively resorbing bone adjacent
to the tumor cells. Panel C shows osteoblastic metastasis
(hematoxylin and eosin, 200). Thickened trabeculae and
large numbers of osteoblasts are next to the bone surface, and there are tumor cells from adenocarcinoma of
the lung between the two large trabeculae. Panel A was
provided by Dr. Hua Zhang, Helen Hayes Hospital, New
York, and Panels B and C were provided by Dr. Brendan
Boyce, University of Rochester, Rochester, New York.

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Osteoclasts arise from precursor cells in the monocytemacrophage lineage12 that differentiate into
inactive osteoclasts. Activated osteoclasts resorb
bone and eventually undergo apoptosis. As shown
in Figure 2A, both locally produced cytokines and
systemic hormones regulate the formation and activity of osteoclasts. The bone microenvironment
plays a critical role in the formation of osteoclasts
through the production of macrophage colonystimulating factor and receptor activator of nuclear
factor-kB (RANK) ligand (RANKL)13,14 by stromal
cells or osteoblasts.
RANKL, a member of the family of tumor necrosis factors, is expressed on the surface of osteoblasts and stromal cells and is released by activated
T cells.13 Most osteotropic factors, such as parathyroid hormone, 1,25-dihydroxyvitamin D3, and prostaglandins, induce the formation of osteoclasts by
increasing the expression of RANKL on marrow
stromal cells and osteoblasts rather than by acting directly on osteoclast precursors15,16 (Fig. 3).
RANKL binds the RANK receptor on osteoclast precursors and induces the formation of osteoclasts by
signaling through the nuclear factor-kB and Jun
N-terminal kinase pathways. A soluble form of
RANKL produced by activated T cells has been detected in the joint fluid of animals with adjuvant arthritis.17
The importance of RANKL in the formation of
osteoclasts has been demonstrated clearly by the
technique of homologous recombination in which
the RANKL or RANK gene has been deleted in mice
(knockout mice). These animals lack osteoclasts,
and as a result, severe osteopetrosis develops.18,19

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mechanisms of disease

Figure 2. Regulation of Bone Resorption (Panel A)

and Bone Formation (Panel B).
Both systemic factors and locally acting factors induce
the formation and activity of osteoclasts (Panel A).
Systemic hormones such as parathyroid hormone,
1,25-dihydroxyvitamin D3, and thyroxine (T4) stimulate
the formation of osteoclasts by inducing the expression
of receptor activator of nuclear factor-kB ligand (RANKL)
on marrow stromal cells and osteoblasts. In addition,
osteoblasts produce interleukin-6, interleukin-1, prostaglandins, and colony-stimulating factors (CSFs), which
induce the formation of osteoclasts. Accessory cells such
as T cells can produce cytokines that can inhibit the formation of osteoclasts, such as interleukin-4, interleukin18, and interferon-g. TGF-b denotes transforming
growth factor b. Plus signs indicate stimulation, and
minus signs inhibition.
Both systemic factors and locally acting factors can enhance the proliferation and differentiation of osteoblasts
(Panel B). These include parathyroid hormone, prostaglandins, and cytokines as well as growth factors such as
platelet-derived growth factor (PDGF) produced by lymphocytes. In addition, bone matrix is a major source of
growth factors, which can enhance the proliferation and
differentiation of osteoblasts. These include the bone
morphogenetic proteins (BMPs), TGF-b, insulin-like
growth factors (IGFs), and fibroblast growth factors
(FGFs). Corticosteroids can induce apoptosis of osteoblasts and block bone formation.

In addition, the development of B cells and T cells

is defective in these animals. A decoy receptor for
RANKL, osteoprotegerin, is normally present in the
bone marrow.20 Osteoprotegerin, a member of the
superfamily of tumor necrosis factor receptors, inhibits the differentiation and resorption of osteoclasts in vitro and in vivo.
The ratio of RANKL to osteoprotegerin regulates
the formation and activity of osteoclasts. Overproduction of osteoprotegerin in transgenic mice causes severe osteopetrosis, whereas the absence of osteoprotegerin results in marked osteopenia.21,22
The importance of RANKL in bone destruction has
led to the development of recombinant osteoprotegerin and antibodies against RANKL as potential
treatments for bone metastasis.
Osteoclasts resorb bone by secreting proteases
that dissolve the matrix and producing acid that releases bone mineral into the extracellular space under the ruffled border of the plasma membrane of
osteoclasts, which faces bone and is the resorbing
organelle of the cell.23 The adherence of osteoclasts
to the bone surface is critical for the bone resorptive
process, since agents that interfere with osteoclast
attachment block bone resorption.24 Agents that af-

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blasts.30 As shown in Figure 2B, many factors can

enhance the growth and differentiation of osteoblasts, including platelet-derived growth factor, fibroblast growth factor, and transforming growth
factor b.31

osteolytic metastasis

Figure 3. Receptor Activator of Nuclear Factor-kB Ligand (RANKL)

and Osteoclast Formation.
RANKL is a potent inducer of osteoclast formation. Osteotropic factors, 1,25dihydroxyvitamin D3, parathyroid hormone (PTH), prostaglandin E-2, and interleukin-1, induce the formation of osteoclasts by up-regulating the expression
of RANKL on the surface of marrow stromal cells and immature osteoblasts.
RANKL then binds its receptor, RANK, on the surface of osteoclast precursors
and signals through the nuclear factor-kB (NF-kB)and Jun N-terminal kinase
(JNK) pathways to induce the formation of osteoclasts and promote osteoclast survival. In addition to RANK, a decoy receptor, osteoprotegerin, inhibits
RANKL binding to RANK. RANKL can also occur in a soluble form produced
by T cells in inflammatory states. The ratio of RANKL to osteoprotegerin determines the level of osteoclastogenesis.

fect the adherence of osteoclasts to bone or inhibit

proteases produced by osteoclasts, such as cathepsin K, are under development and may be useful for
treating bone metastases.
osteoblasts

Osteoblasts are the bone-forming cells. They arise

from mesenchymal stem cells, which form osteoblasts, adipocytes, and muscle cells.25 A transcription factor that is critical for the differentiation of
osteoblasts is Runx-2, or core-binding factor a1
(CBFA1). CBFA1 drives the expression of most
genes associated with osteoblast differentiation.26
Bone does not develop in mice that lack the CBFA1
gene.27,28 The differentiation of osteoblasts is less
well understood than the differentiation of osteoclasts. It is clear that there is an early osteoblast
precursor that produces alkaline phosphatase and
a more differentiated precursor that produces increasing amounts of osteocalcin and calcified matrix.29 Osteoblasts eventually become osteocytes.
Bone morphometric proteins are critical factors that
stimulate the growth and differentiation of osteo-

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In osteolytic metastases, the destruction of bone is

mediated by osteoclasts rather than tumor cells.32,33
However, the factors responsible for the activation
of osteoclasts vary depending on the tumor. In multiple myeloma, osteoclasts accumulate only at boneresorbing surfaces adjacent to myeloma cells; their
levels are not increased in areas uninvolved with tumor.34 In addition to the increase in bone resorption, bone formation is suppressed so that bone
lesions in patients with myeloma become purely
lytic.35
Several osteoclastogenic factors have been implicated in the increased activity of osteoclasts in myeloma.36 The leading candidates are interleukin-1,
interleukin-6, macrophage inflammatory protein
1a, and RANKL. Interleukin-1 is a potent stimulant
of osteoclast formation,37 but levels of interleukin-1
produced by myeloma cells are extremely low.38 Sati
et al.39 and Soutar et al.40 did not detect significantly increased levels of interleukin-1b in myeloma samples, suggesting that interleukin-1 may not
be a major mediator of myeloma bone disease.
Interleukin-6 is a growth factor or at least blocks
apoptosis of myeloma cells.41 It is present in marrow plasma samples from patients with myeloma.42
Interleukin-6 is a potent stimulator of osteoclast
formation43 and can enhance the effects of parathyroid hormonerelated peptide on the formation
of osteoclasts in vivo.44 Interleukin-6 levels in the
marrow have not consistently been correlated with
the presence of bone lesions, however.45,46 When
myeloma cells adhere to marrow stromal cells, the
production of interleukin-6 by marrow stromal cells
increases.47 Thus, interleukin-6 appears to have an
important role in enhancing the growth or prolonging the survival of myeloma cells, but its role in myeloma bone disease remains to be determined.
RANKL is a major mediator of myeloma bone
disease. Several studies have suggested that myeloma cells produce RANKL,48,49 but it is unclear
whether the amount of RANKL produced by myeloma cells is sufficient to induce the formation of
osteoclasts. Instead, it may simply prevent apoptosis of osteoclasts. RANKL is produced by marrow

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mechanisms of disease

stromal cells in myeloma. In the microenvironment of bone in myeloma, RANKL production is

increased and osteoprotegerin is markedly decreased.50 Blocking the binding of RANKL to the
RANK receptor with a soluble form of the RANK
receptor or osteoprotegerin inhibits bone destruction in a mouse model of myeloma.51,52 All these
data suggest that RANKL is a major mediator of
myeloma bone disease.
Macrophage inflammatory protein 1a also appears to be a key regulator of bone destruction in
myeloma.53,54 Macrophage inflammatory protein
1a is a potent inducer of osteoclast formation in vitro, independently of RANKL, and enhances both
RANKL-stimulated and interleukin-6stimulated
osteoclast formation.55 In approximately 70 percent
of patients, myeloma cells produce macrophage inflammatory protein 1a, and the level of this protein
is elevated in bone marrow plasma.53 Macrophage
inflammatory protein 1a levels correlate strongly
with the presence of osteolytic lesions; moreover,
DNA microanalysis of myeloma cells has shown
that the expression of the macrophage inflammatory protein 1a gene is markedly increased and correlates with bone disease.56 Furthermore, blocking
expression of the gene for macrophage inflammatory protein 1a or the activity of macrophage inflammatory protein 1a in murine models of myeloma decreases both bone destruction and myeloma
tumor burden.57,58 Macrophage inflammatory protein 1a also enhances adhesive interactions between
myeloma cells and stromal cells by up-regulating
the expression of b1 integrins on myeloma cells.57
Adhesive interactions between marrow stromal cells
and myeloma cells increase the production of interleukin-6, RANKL, and macrophage inflammatory
protein 1a, further increasing bone destruction.

osteoblast dysfunction
in myeloma
Bone lesions in myeloma are purely lytic there is
no osteoblastic response. This phenomenon explains the clinical observation that in about half the
cases of myeloma, bone scans are normal in the
presence of severe osteolytic bone destruction.59
The basis of the decreased osteoblastic response in
myeloma is unknown. Myeloma cells can produce
tumor necrosis factor a, which inhibits osteoblastic
growth and differentiation.60 However, tumor necrosis factor a has not been implicated in the suppression of bone formation in myeloma. Although

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cocultures of two interleukin-6dependent myeloma cell lines with osteoblast-like human osteosarcoma cells reduced the amounts of osteocalcin produced by the cells,61 the identity of the factor or
factors responsible is unknown.
Recently, Tian and coworkers,62 using genemicroarray analysis and immunohistochemical
analysis, found that myeloma cells expressed dickkopf 1 (DKK1), a Wnt-signaling antagonist, and
that the presence of high levels of DKK1 correlated
with focal bone lesions in patients with myeloma.
They further demonstrated that bone marrow serum
from these patients that contained more than 12 ng
of DKK1 per milliliter inhibited osteoblastic differentiation in a murine myoblast cell line. These data
suggest that DKK1 may be involved in the inhibition
of osteoblast differentiation in myeloma. It is likely
that more than one factor is involved in the suppression of osteoblast activity in myeloma; this situation is analogous to the multiplicity of factors that
increase osteoclast activity.

osteolytic metastasis
from breast cancer
the vicious circle
Tumor cells in breast cancer produce factors that
directly or indirectly induce the formation of osteoclasts. In turn, bone resorption by osteoclasts releases growth factors from the bone matrix that stimulate tumor growth and bone destruction.63 This
reciprocal interaction between breast-cancer cells
and the bone microenvironment results in a vicious
circle that increases both bone destruction and the
tumor burden (Fig. 4).
Bone is an abundant source of inactive growth
factors, which are activated during the bone-resorptive process10 and which can then stimulate the
growth of breast-cancer cells. Parathyroid hormonerelated peptide is probably the factor produced by breast-cancer cells and most solid tumors
that stimulates the formation of osteoclasts.64,65
Both parathyroid hormonerelated peptide and
parathyroid hormone bind the same receptor
(PTHR1) and induce the expression of RANKL on
marrow stromal cells. Parathyroid hormone is the
main peptide regulator of calcium homeostasis, and
parathyroid hormonerelated peptide has biologic
effects on bone similar to those of parathyroid hormone.66 In the amino acid sequences of parathyroid hormone and parathyroid hormonerelated
peptide, 8 of the first 13 amino acids are identical,

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Figure 4. The Vicious Circle of Osteolytic Metastasis.

Tumor cells, in particular breast-cancer cells, secrete
parathyroid hormonerelated peptide as the primary
stimulator of osteoclastogenesis. In addition, tumor
cells produce other factors that increase the formation
of osteoclasts, including interleukin-6, prostaglandin E2
(PGE2), tumor necrosis factor, and macrophage colonystimulating factor (M-CSF). These factors increase the
expression of receptor activator of nuclear factor-kB ligand (RANKL), which directly acts on osteoclast precursors to induce the formation of osteoclasts and bone
resorption. The process of bone resorption releases factors such as transforming growth factor b (TGF-b), insulin-like growth factors (IGFs), fibroblast growth factors
(FGFs), platelet-derived growth factor (PDGF), and bone
morphogenetic proteins (BMPs), which increase the
production of parathyroid hormonerelated peptide by
tumor cells as well as growth factors that increase tumor
growth. This symbiotic relationship between bone destruction and tumor growth further increases bone
destruction and tumor growth.

and both peptides have similar three-dimensional

structures.66
The production of parathyroid hormonerelated
peptide is increased in metastases of breast cancer
to bone. Only 50 percent of primary breast cancers express parathyroid hormonerelated peptide,
whereas 92 percent of metastases of breast cancer
to bone produce the peptide.67 However, it is unclear whether this difference results from induction
of the peptide in the bone microenvironment or
whether tumors that produce the peptide are more
likely to metastasize to bone. When breast-cancer
cells from patients are injected into nude mice and
metastasize to bone, they increase the production

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of parathyroid hormonerelated peptide.64 The peptide induces the formation of osteoclasts and bone
resorption, which releases transforming growth
factor b. Transforming growth factor b, in turn,
further increases production of the peptide by the
breast-cancer cells.68 An antibody against parathyroid hormonerelated peptide is being evaluated in
patients with bone metastases from breast cancer.
In the vicious circle of breast-cancer metastases
(Fig. 4), bone destruction increases local calcium
levels, which promotes tumor growth and the production of parathyroid hormonerelated peptide.69
Breast-cancer cells also produce, or induce, interleukin-6, prostaglandin E2, macrophage colony-stimulating factor, interleukin-1, and tumor necrosis
factor a,70,71 which may also play an important role
in the induction of osteoclast formation by breastcancer metastases. Prostaglandin E2 can increase
the expression of RANKL and directly enhance the
effects of RANKL on the formation of osteoclasts.71
Together, these data suggest that parathyroid hormonerelated peptide is a major mediator of osteolytic bone destruction by breast cancer and other
solid tumors.

therapeutic implications
of resorption and tumor
growth
There is a close relationship between bone destruction and tumor growth. For example, treating myeloma in mice with agents that block bone resorption but have no direct effect on tumor growth not
only inhibits the formation of osteoclasts but also
decreases the tumor burden.51,52 However, definitive evidence that decreasing bone destruction decreases the tumor burden in patients with bone
metastasis is lacking. Gordon and coworkers72 reported that a single infusion of a potent bisphosphonate that blocks bone resorption markedly increased apoptosis of myeloma cells in vivo in 10
of 16 patients with newly diagnosed myeloma. In
a large, randomized, double-blind, placebo-controlled study73 of the bisphosphonate clodronate,
Powles et al. found that administration of the drug
was associated with a decrease in both the incidence
of bone metastasis and the death rate in patients
with breast cancer who were at high risk for bone
metastasis. However, blocking bone destruction
does not appear to affect the growth of tumors in
soft tissues,73 indicating the unique characteristics
of tumor growth in bone.

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mechanisms of disease

osteoblastic metastasis
another vicious circle?
The mechanisms of osteoblastic metastasis and the
factors involved are unknown. Endothelin-1 has
been implicated in osteoblastic metastasis from
breast cancer.74 It stimulates the formation of bone
and the proliferation of osteoblasts in bone organ
cultures,75 and serum endothelin-1 levels are increased in patients with osteoblastic metastasis
from prostate cancer.76 Furthermore, in an animal
model of osteoblastic metastasis, treatment with a
selective endothelin-1Areceptor antagonist decreased both osteoblastic metastasis and the tumor burden.74 The antagonist had no effect on the
growth of the tumor at orthotopic sites. These results suggest that blocking osteoblast-inducing activity by tumors may decrease tumor growth and
osteoblast activity and suggest that a vicious circle
may also be involved in osteoblastic metastasis in
which tumors induce osteoblast activity and thus
the subsequent release from the osteoblasts of
growth factors that increase tumor growth. In addition to endothelin-1, platelet-derived growth factor,77 a polypeptide produced by osteoblasts in the
bone microenvironment, urokinase,78,79 and prostate-specific antigen (PSA)80 may also be involved.

osteoblastic metastasis
in prostate cancer
Overproduction of urokinase-type plasminogen activator (u-PA) by prostate-cancer cells increases
bone metastasis,78 and cells transfected with an
anti-sense DNA to u-PA had one third as many metastases as did cells transfected with an empty vector. Human PC3 prostate-cancer cells produce a factor that is homologous to u-PA.79 Prostate-cancer
cells also release PSA, a kallikrein serine protease.
PSA can cleave parathyroid hormonerelated peptide at the N-terminal, which could block tumorinduced bone resorption. It may also activate osteoblastic growth factors released in the bone
microenvironment during the development of bone
metastases, such as insulin-like growth factors I and
II or transforming growth factor b.80 These data
suggest that a vicious circle may also be responsible for osteoblastic metastasis.
Bone metastases in prostate cancer are predominantly osteoblastic, with increased numbers
of irregular bone trabeculae.81 However, markers of
bone resorption are also increased in metastatic

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prostate cancer, although there is usually no histologic evidence of increased numbers of osteoclasts.
In prostate cancer, levels of bone-resorption markers are higher in patients with bone metastasis than
in patients without bone metastasis and reflect the
extent of bone metastasis more accurately than does
the PSA level.82
Recent clinical trials have suggested that blocking osteoclastic bone resorption decreases related
skeletal events in patients with prostate cancer.6
However, in a murine model of prostate cancer, the
inhibition of osteoclast activity did not inhibit the
development of osteoblastic metastasis.83 Thus, it
is unclear whether bone destruction precedes the
development of osteoblastic metastasis or is a consequence of the increased bone formation. Yi et al.
have shown in an animal model of osteoblastic metastasis that an initial phase of bone destruction is
followed by extensive formation of bone.77 Their
data suggest that bone resorption precedes bone
formation in the development of osteoblastic metastases and that osteoclast activation plays an important role in the development of osteoblastic metastases.

biochemical markers of bone

turnover for the detection
of bone metastasis
The value of biochemical markers of bone turnover
as a means of monitoring patients with bone metastases is still under investigation. Levels of bone-specific alkaline phosphatase, osteocalcin, and type I
procollagen C-propeptide in serum are indicators of
osteoblast activity, whereas serum levels of C-terminal telopeptide of type I collagen and tartrate-resistant acid phosphatase and urinary levels of type I
collagen cross-linked N-telopeptides are markers
of osteoclast activity. All these markers have been
used to assess the response to therapy or for the detection of bone metastases,84-86 but results have
been variable. Urinary type I collagen cross-linked
N-telopeptides and C-terminal telopeptide of type I
collagen appear to be the most useful.84
In summary, bone metastases are among the
most debilitating problems in patients with cancer.
The molecular mechanisms responsible for both osteolytic and osteoblastic metastases are just being
identified. The use of gene arrays and proteomics
and the availability of appropriate animal models of
bone metastasis have permitted the identification

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of factors produced by the tumor cells themselves

or by the microenvironment in response to the tumor that mediate the process of bone destruction.
These types of studies should result in the development of therapeutic agents to treat and possibly
prevent this devastating complication of cancer.

of

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Supported by research funds from the Multiple Myeloma Research Foundation; a Department of Veterans Affairs Merit Review
Award; and grants (AR41336 and AG13625) from the National Institutes of Health.
I am indebted to members of the General Clinical Research Center, University of Pittsburgh Medical Center, for their assistance in
the care of my patients.

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