Download Molecular Pathways: Osteoclast

Published OnlineFirst October 26, 2011; DOI: 10.1158/1078-0432.CCR-10-2507
Clinical
Cancer
Research
Molecular Pathways
Molecular Pathways: Osteoclast-Dependent and
Osteoclast-Independent Roles of the RANKL/RANK/OPG
Pathway in Tumorigenesis and Metastasis
William C. Dougall
Abstract
Receptor activator of nuclear factor-kappa B ligand (RANKL) is a TNF ligand superfamily member that is
essential for the formation, activation, and function of osteoclasts. RANKL functions via its cognate receptor
RANK, and it is inhibited by the soluble decoy receptor osteoprotegerin (OPG). In skeletal metastases, the
ratio of RANKL to OPG is upregulated, which leads to increased osteoclast-mediated bone destruction. These
changes in the bone microenvironment not only compromise the structural integrity of bone, leading to
severe clinical morbidities, but have also been implicated in establishment of de novo bone metastasis and
the progression of existing skeletal tumors. Evaluation of RANKL inhibitors, including the fully human antiRANKL antibody denosumab, in patients with cancer has shown reductions in tumor-induced bone
resorption activity and successful management of skeletal complications of bone metastases. RANKL also
functions as a major paracrine effector of the mitogenic action of progesterone in mouse mammary
epithelium, and it has a role in ovarian hormone-dependent expansion and regenerative potential of
mammary stem cells. RANKL inhibition attenuates mammary tumorigenesis and pulmonary metastases in
mouse models. These data suggest that the contribution of progesterone to increased mammary cancer
incidence is mediated, at least in part, by RANKL-dependent changes in the mammary epithelium; RANKL
also directly promotes distant metastases. In summary, the antitumor and antimetastatic effects of RANKL
inhibition can occur by at least 2 distinct mechanisms, one in the bone via osteoclast-dependent effects, and
the second via direct effects on the tumor cells of various origins and/or mammary epithelium. Clin Cancer
Res; 18(2); 326–35. 2011 AACR.
Background
Control of normal bone remodeling by the
RANK/RANKL pathway
The cells that control bone remodeling include the osteoblast, which deposits new bone, and the osteoclast, a
specialized cell of hematopoietic origin that resorbs the
inorganic and organic matrix of the bone. Osteoblast and
osteoclast function are coordinately regulated in normal
bone remodeling. Identification of the receptor activator of
nuclear factor-kappa B (RANK), RANK ligand (RANKL),
and osteoprotegerin (OPG) pathway in the mid-1990s
revealed a key molecular axis for osteoclast formation,
function, and survival, and it provided crucial insights into
the regulation of normal, physiologic bone remodeling.
Functional genomics in mice showed that RANKL
Author's Affiliation: Department of Hematology and Oncology Research,
Amgen Inc., Seattle, Washington
Corresponding Author: William C. Dougall, Department of Hematology and Oncology Research, Amgen Inc., 1201 Amgen Ct. West,
Seattle, WA 98119. Phone: 206-265-7553; Fax: 206-217-0494; E-mail:
[email protected]
doi: 10.1158/1078-0432.CCR-10-2507
2011 American Association for Cancer Research.
326
(TNFSF11), a member of the TNF ligand superfamily, and
RANK (TNFRSF11a), the cognate TNFR family receptor for
RANKL, were essential for osteoclastogenesis in vivo. A
functional interaction between RANKL, expressed by bone
stromal cells of the osteoblast lineage, and RANK, expressed
by osteoclast precursors of hematopoietic myeloid lineage,
is necessary for osteoclast differentiation, survival, and
activation. Knockout mice lacking either RANK or RANKL
develop significant osteopetrosis resulting from a lack of
osteoclasts and absence of bone resorption (1, 2). OPG
(TNFRSF11b), another member of the TNFR superfamily,
can also bind RANKL and functions as a soluble decoy
receptor for RANKL. The critical role of OPG in osteoclastogenesis and bone remodeling was first shown by the
increased bone mass as a result of reduced osteoclast numbers observed in transgenic mice overexpressing OPG (3)
and, conversely, the osteopenia observed in OPG knockout
mice (4). Given that the precise and balanced interaction of
RANK, RANKL, and OPG is critical for osteoclastogenesis
and, therefore, the maintenance of homeostatic bone remodeling and bone mass, it was hypothesized that this pathway may become dysregulated within the bone during
disease states and contribute to pathologic bone loss, such
as postmenopausal osteoporosis or cancer-induced bone
destruction (Fig. 1).
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RANKL in Tumorigenesis and Metastasis
A
Physiological bone
turnover/remodeling (normal)
B
Tumor-induced pathologic
bone turnover/remodeling (high)
Tumor
7
Tumor, dormant
micro-metastasis
Variety of
tumor-derived
factors
1
Bone
stromal
cell
Bone
stromal
cell
RANKL
3
Osteoclast
Osteoblast
Bone resorption
Bone formation
Expanded
tumor mass
6
2 Tumor cell
expressing
RANKL
RANKL
5
↑ RANKL
↓ OPG
Osteoclasts
Osteoblasts
↑ Bone formation
↑ Bone resorption
4
Bone destruction
(spectrum of lesions)
Homeostatic
maintenance of
bone mass
Osteoblastic
Mixed
osteolytic/
osteoblastic
Osteolytic
© 2011 American Association for Cancer Research
Figure 1. Bone-intrinsic functionality of the RANKL pathway in normal and pathologic osteoclastogenesis. A, normal bone turnover and/or
remodeling observed in healthy physiologic systems. Bone stromal cells, including cells of the osteoblast lineage, provide a limited amount of RANKL, which
leads to osteoclast differentiation, survival, and activation and subsequent bone resorption. Resorption is balanced by osteoblast-dependent new bone
formation. If occult micrometastases were to exist, they would remain dormant because of the low level of bone turnover (37). B, tumor-induced high
bone turnover and/or remodeling. (1) A variety of tumor-derived factors will cause increases in RANKL and/or decreases in OPG within the bone stroma. (2)
Some tumor cells can also directly produce RANKL. (3) The increased RANKL-to-OPG ratio leads to increased osteoclast formation, survival, and
activity, which increases the rate of bone remodeling and/or turnover. (4) Pathologic bone remodeling, characterized by increased osteoclast and osteoblast
activities, causes a spectrum of bone lesions in patients with bone metastasis, ranging from predominately osteoblastic to predominately osteolytic. Multiple
myeloma gives rise to purely lytic bone lesions. (5) As a result of the increased osteoclast function, many changes occur within the reactive bone
microenvironment. These changes include increases in local levels of calcium, release of activated growth factors from the bone matrix, and increased
production of growth and/or angiogenic factors by the osteoclast. (6) Increased tumor cell growth and survival, including the outgrowth of dormant
micrometastases, occur as a result of these bone matrix–and osteoclast-derived factors. (7) Skeletal tumor cells respond to these bone signals with further
production of additional proresorptive factors, generating a feed-forward loop known as the "vicious cycle."
A unique feature of skeletal metastases and multiple
myeloma is the active involvement of the host, particularly osteoclasts, in the pathophysiologic process leading
to bone destruction and skeletal tumor growth. Accelerated osteoclast action and subsequent increased bone
resorption have been operatively linked to a full spectrum
of skeletal complications (e.g., hypercalcemia, pathologic
fracture, bone pain, spinal cord compression, surgery or
radiation treatment to the bone, etc.) that result from
bone metastases and multiple myeloma. Osteoclast
involvement is observed in all types of cancer-induced
bone destruction, regardless of the lesions’ radiographic
appearance as either osteolytic or osteoblastic, and irrespective of the tumor type of origin (5–7).
In addition to causing destructive bone loss and associated clinical sequelae, tumor-induced osteoclasts contribute to the establishment, growth, and survival of tumors.
www.aacrjournals.org
Products of osteoclastic bone resorption, including matrixderived growth factors [e.g., TGF-b, insulin-like growth
factor I (IGF-I)] and elevated calcium levels, increase tumor
cell proliferation and survival and induce further production of osteolytic and osteoblastic factors, thereby creating a
positive feedback loop known as the "vicious cycle" (7). As
osteoclasts are highly related to macrophages, it is perhaps
not surprising that many protumorigenic factors can be
produced by osteoclasts directly, including growth factors
[platelet-derived growth factor a, interleukin 1 (IL-1), TNF,
IL-6, IGF-I, BAFF, APRIL] or angiogenic factors [VEGFa,
VEGFc, basic fibroblast growth factor (bFGF)], in addition
to the production of matrix metalloproteinases (e.g.,
MMP-7, MMP-9, MMP-14; refs. 8, 9). Thus, the osteoclast
may indirectly provide factors that contribute to skeletal
tumor establishment and progression, as a consequence of
bone matrix turnover, in addition to cellular production,
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Dougall
independent of bone resorption. Initial studies of breast
cancer by Stephen Paget in 1889 identified the bone as an
optimal site (or "soil") for distant metastases ("seed";
ref. 10). It is now clear that many other tumors, including
prostate, lung, renal, melanoma, and thyroid, have a high
propensity to metastasize to bone, highlighting the skeleton
as a fecund metastatic site capable of harboring early tumor
colonization and actively promoting the growth of skeletal
tumors.
The RANKL, RANK, OPG pathway in bone- and
cancer-induced bone disease
In each of these aforementioned pathologic processes
(bone destruction, metastatic establishment, and tumor
progression), the tumor has coopted the host by upsetting
the balance of 2 key factors that normally govern physiologic osteoclast formation and bone remodeling: RANKL
and OPG (Fig. 1). The cancer-induced signals capable of
altering the normally well-balanced RANKL-to-OPG ratio
can be extremely diverse, reflecting the different sources of
RANKL and OPG, the assortment of tumor types that affect
bone, and the heterogeneity of individual tumor types
(7, 11). More importantly, these diverse signals and
mechanisms that elevate osteoclastogenesis and bone
destruction converge on the RANKL pathway. Many different cytokines or factors produced by skeletal tumors [e.g.,
IL-1b, IL-6, IL-8, IL-11, IL-17, macrophage inflammatory
protein 1a, TNFa, parathyroid hormone-releasing protein
(PTHrP), prostaglandin E (PGE2)] cause increased RANKL
production by stromal cells in the bone microenvironment,
including cells of the osteoblast lineage. OPG, the normal
decoy receptor for RANKL, can also be downregulated by
tumors via various mechanisms; reduced synthesis or active
degradation of OPG in the bone is observed as a consequence of tumor-derived factors (11). Certain factors in
metastatic cancer have dual effects on the RANKL-to-OPG
ratio. For instance, PTHrP, IL-1, and PGE2 have been shown
to act on the bone stroma and stimulate osteoclast activity
by increasing RANKL and decreasing OPG simultaneously
(7). T lymphocytes, including activated T cells (12) and
CD4þCD25þ T-regulatory cells (T-reg; ref. 13), may be
another source of RANKL in bone metastases or other
extraskeletal cancer settings in which RANKL may function
(see below). Multiple myeloma cells can increase RANKL
production in T lymphocytes (14); however, functional
evidence for T-cell contribution of RANKL in cancer–bone
interactions is lacking, largely because most preclinical
bone cancer models are studied in immunocompromised
hosts.
Increases in RANKL that lead to cancer-induced osteoclastogenesis are not limited to infiltrating immune cells or
reactive changes in the bone stroma caused by the tumor but
can be contributed by the tumor cells themselves. RANKL
expression has also been reported on some tumor types,
including breast cancer, prostate cancer, multiple myeloma,
and renal carcinoma (as described below). RANKL produced by tumor cells can increase osteoclastogenesis in vitro
(15), suggesting that the tumor cell localized in the skeleton
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Clin Cancer Res; 18(2) January 15, 2012
may also directly contribute an osteoclastogenic signal. It is
certainly possible that the bone microenvironment provides signals that can increase RANKL on metastatic tumor
cells, which in turn enhances further osteoclast activity and
creates a feed-forward loop. For instance, RANKL increases
have been observed in prostate tumor cell lines after treatment with TGF-b (16), a product of bone resorption, or
upon stimulation of the epithelial–mesenchymal transition
(EMT) by TGF-b plus epidermal growth factor treatment or
transfection with SNAIL (17). Intriguingly, EMT changes in
tumor cells residing in the bone have been linked to a more
invasive phenotype, and development of overt bone metastases from dormant micrometastases (17, 18), although a
functional role for RANKL in this transition, has not yet
been defined. Consistent with the hypothesis that RANKL
production by tumor cells accelerates osteoclast formation
and bone metastasis is the observation that a high expression level of RANKL in primary renal cell carcinoma is
associated with a shorter bone metastasis–free survival
(19). A similar relationship between high RANKL expression levels in primary tumors and development of bone
metastases has also been described for patients with hepatocellular carcinoma (20).
Currently, no definitive data correlate circulating levels of
RANKL in serum with bone metastases. This lack of data
may reflect the preponderance of the membrane-bound
versus soluble form of RANKL, the relatively low levels of
RANKL detected in the serum, or the technical limitations of
such assays (21). It is also likely that increases in RANKL
remain localized to the bone lesion, where focal activation
of bone remodeling and increased osteoclast activity are
closely juxtaposed to tumor infiltration (22). In contrast, a
relationship between the higher ratio of serum RANKL to
OPG and a greater extent of bone lesions has been observed
in multiple myeloma (23), which may reflect either the
extensive tumor cell production of RANKL or the systemic
nature of this hematologic tumor.
To address the functional contribution of RANKL to
cancer-induced bone disease, pharmacologic RANKL inhibitors, such as OPG and RANK-Fc, have been tested in
rodent models, and these studies have been the subject of
recent reviews (11). Preclinical models of cancer bone
metastasis and multiple myeloma typically develop osteolytic, osteoblastic, or mixed lesions after systemic or intratibial injection in rodents. RANKL inhibition has been
tested in models representing many different tumor types,
including multiple myeloma, breast cancer, prostate cancer,
lung cancer, colon cancer, etc., and in each case has been
shown to effectively reduce tumor-induced bone lesions
(11). Given the specific mechanism of RANKL inhibition,
the observed broad activity across lesion types suggests that
osteoclastic activity may be a requisite element for both
osteolytic and osteoblastic lesions. In addition to the beneficial effect on bone lesions, RANKL blockade has also
reduced other tumor-associated sequelae, including bone
pain (24) and hypercalcemia of malignancy (25).
On the basis of the observed reciprocal feedback (the
vicious cycle) occurring between bone and tumor, one
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RANKL in Tumorigenesis and Metastasis
would predict that reduction of bone resorption would not
only reduce the tumor-induced bone destruction but also
slow skeletal tumor progression. Studies using preclinical
cancer–bone models representing different solid tumors
and multiple myeloma have shown a substantial reduction
in tumor growth in the bone as a consequence of the potent
osteoclast blockade achieved with RANKL inhibition (6,
7, 26). The reductions in skeletal tumor burden upon
RANKL inhibition are dependent on the bone microenvironment and are associated with increased tumor cell
apoptosis, decreased tumor cell proliferation, and increased
survival of tumor-bearing mice (27–30). RANKL inhibition
effectively reduces skeletal tumor growth as a monotherapy,
but as would be predicted by an approach that targets the
bone microenvironment and reduces local growth factor
and calcium production, the reduction in skeletal tumor
burden is additive when combined with chemotherapy,
hormonal therapy, or targeted therapies (28, 31–33).
If the "seed and soil" observations and "vicious cycle"
hypothesis postulate that the fertile nature of the bone
microenvironment actively contributes to the processes of
early tumor colonization and metastatic outgrowth, then
skeletal metastases might be prevented or delayed through
pharmacologic blockade of bone turnover (34), with
RANKL inhibition as a potential approach. To test this
hypothesis in preclinical models, however, one must consider limitations of experimental bone metastases models,
including the observation that skeletal growth of xenografted human tumor cells may ultimately achieve a critical
mass capable of self-maintenance independent of the bone
microenvironment. To address this limitation, it has been
necessary to model tumor–bone interactions at early stages
of bone metastasis and colonization using high-sensitivity,
small-animal imaging techniques. Prophylactic treatment
of mice with the RANKL inhibitor OPG-Fc was employed to
reduce baseline osteoclast activity and bone resorption
prior to inoculation of breast tumor MDA-231 cells. This
strategy subsequently delayed de novo formation of metastatic skeletal tumors as monitored by bioluminescent
imaging, presumably by arresting the vicious cycle supporting initial tumor growth (27).
The RANKL pathway in normal mammary biology and
cancer
In addition to severe osteopetrosis, RANK and RANKL
knockout mice manifested a lactation defect, revealing an
intrinsic functionality of the pathway in mammary epithelium that is also relevant to mammary tumorigenesis and
metastases. The failure of RANK or RANKL knockout mice
to lactate was due to a marked inability to develop lobuloalveolar mammary structures, which normally undergo a
massive expansion under hormonal control during pregnancy and eventually differentiate into milk-secreting tissue. Proliferation and survival of the mammary epithelium
are reduced in the absence of a productive RANKL/RANK
signal, and transplantation experiments showed this defect
to be autonomous to mammary epithelial tissue (35).
RANKL expression is greatly increased in luminal mammary
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epithelial cells at midgestation pregnancy and can be
induced by factors such as prolactin, PTHrP, and progesterone (35). In mice, RANK expression is observed at low
levels in luminal and basal cells of the mammary epithelium and becomes more highly expressed at midgestation
pregnancy, most selectively at ductal branch points (36).
Genetic studies revealed that both RANKL and progesterone receptor (PR) function at similar stages in lactational
morphogenesis and that these proteins are colocalized in
luminal mammary epithelial cells. Recent experiments have
shown that the major mitogenic effect of progesterone
occurs via increases in RANKL within PRþ luminal epithelial
cells. RANKL, acting as a paracrine factor, then induces
mitogenesis of neighboring estrogen receptor (ER)/PR
mammary epithelial cells (37–39). Moreover, other recent
studies have shown that RANKL can mediate the nonproliferative expansion of the mouse mammary gland via
increases in the number and regenerative capacity of mammary stem cells (MaSC). Despite their ER/PR phenotype,
MaSC are profoundly responsive to ovarian hormone signaling, and RANKL was identified as a paracrine effector of
the progesterone-dependent effects on MaSC occurring
during either gestation or estrous cycles (40, 41).
RANKL promotes mammary tumorigenesis
The fundamental roles of the RANKL pathway in the
normal physiology of the mammary gland have significant
implications in cancer and tumorigenesis. RANKL-driven
hormone (progesterone)-dependent proliferation, survival,
and nonproliferative expansion of MaSC could each contribute to mammary cancer initiation, progression, and
recurrence (Fig. 2). During normal lactational morphogenesis, it is well characterized that RANK and RANKL adhere to
strict, yet overlapping, spatial–temporal expression patterns
within the mammary epithelium under control of lactation
hormones. However, when RANKL or RANK is overexpressed in the mammary epithelium [via mouse transgenic
models using the mouse mammary tumor virus (MMTV)
promoter] in the absence of strict hormonal control, inappropriate mammary proliferation is observed (36, 42).
Despite the aberrant proliferation (including hyperplasia)
induced by an overactive RANKL pathway in these 2 models, spontaneous mammary tumors were not observed in
aged virgin mice. However, accelerated preneoplasias and
increased mammary tumor formation were observed in
MMTV-RANK mice after multiparity or treatment with
the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA)
in combination with a synthetic progestin hormone
[medroxyprogesterone acetate (MPA); ref. 38], settings in
which mammary epithelial RANKL expression is increased
owing to increased sex hormone levels. Pharmacologic
blockade of RANKL decreased incidence and delayed onset
of mammary tumors induced by DMBA and MPA in both
MMTV-RANK transgenic mice and wild-type mice (38). The
reduction in mammary tumor formation was preceded by a
reduction in preneoplasias and correlated with rapid and
sustained reductions in hormone-induced mammary epithelial proliferation and cyclin D1 expression, as well as
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Potential sources
of RANKL
Dougall
RANKL by
hormones
(Progesterone,
PTHrP, Prolactin)
RANKL in
preneoplastic or
neoplastic cells
RANKL
in T-cells
RANKL-induced effects
in tumorigenesis and
metastasis
RANKL at metastatic
sites (lymph node, bone)
Bone
Mammary
stem cell
(MaSC)
Lung
Normal
mammary
epithelium
Preneoplasia
Adenocarcinoma
Metastasis
Mechanism of RANKLdependent effects
(in vivo and in vitro)
Proliferation
Survival
Loss of apical/basal polarity
Migration/
invasion
Anchorageindependent growth
Expansion and
regenerative capacity
of stem cell component
© 2011 American Association for Cancer Research
Figure 2. Direct protumorigenic and prometastatic activities of RANKL. Using mammary transformation as a model, several different sources of RANKL are
indicated, which may impact multiple steps in tumor progression. For instance, hormonal influences (e.g., progesterone, PTHrP, prolactin) may increase
RANKL not only in the normal mammary epithelium but also in preneoplastic, tumor, and metastatic lesions. RANKL has also been observed within
preneoplastic and neoplastic cells independent of hormone influences. RANKL expressed on T lymphocytes may potentially be important at multiple stages in
cancer, from preneoplasias through metastases. Certain metastatic sites, such as the lymph node and bone, are rich sources of RANKL. Different RANKLdependent activities relevant to cancer formation and progression are summarized above. RANKL may function at multiple steps in tumor initiation,
progression, and recurrence, including the increased proliferation and survival of normal mammary epithelium and tumor cells, as well as the enhanced
numbers and regenerative potential of MaSC or the putative stem cell component of tumors. Other RANKL-dependent activities on tumor cells potentially
relevant to distant metastatic spread are reflected by the in vitro observations of enhanced migration and invasion or increased growth in a semisolid medium.
increased apoptosis, suggesting that RANKL affects early
stages of tumor formation. A similar reduction in mammary
tumor formation (also induced by MPA and/or DMBA) was
observed by Schramek and colleagues (43) in mice, in
which RANK had been selectively deleted from the mammary epithelium by a tissue-specific Cre recombinase–
mediated approach. This genetic approach also revealed
that the RANKL pathway protects against DNA damageinduced cell death and expands the putative stem cell
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Clin Cancer Res; 18(2) January 15, 2012
component of mammary tumors. In a model of mammary
cancer induced by the c-neu oncogene in the absence of
exogenous hormone (MMTV-neu transgenic mice), inhibition of RANKL (beginning at 5 months of age) did not affect
median time to spontaneous mammary tumor formation,
but it did decrease the number of preneoplastic lesions and
mammary tumors (38). Mammary tumor formation was
not reduced with another inhibitor of bone resorption [i.e.,
the nitrogen-containing bisphosphonate zoledronic acid
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RANKL in Tumorigenesis and Metastasis
(ZA); ref. 38]. In line with the observed reductions in
mammary epithelial biomarkers upon RANKL inhibition
and the protection against tumors observed in the mammary-selective knockout of RANK, these data confirm direct
osteoclast-independent functionality of the RANKL pathway in cancer.
A direct role of the RANKL pathway in distant
metastases
There is also preclinical evidence that blockade of RANKL
will reduce distant metastasis, potentially via mechanisms
distinct from the bone-intrinsic antiosteoclast effect (Fig. 2).
Treatment of transgenic MMTV-neu mice with RANK-Fc
significantly decreased spontaneous lung metastases (38).
Consistent with this observation, crossing the MMTV-neu
mice with RANK heterozygotes reduced the incidence of
spontaneous lung metastases (44). Reductions in bone
metastases or lung metastases have been achieved with
pharmacologic blockade of RANKL in animals bearing
RANK-positive melanoma cells (45) or transplanted primary MMTV-neu tumor cells (44). Reciprocally, enhanced lung
metastases have been observed upon systemic RANKL exposure in mice bearing orthotopically transplanted tumor
cells, originating from either a RANK-positive human breast
tumor cell line or tumor cells derived from MMTV-neu mice
(44).
To gain mechanistic insight into the prometastatic activity of RANKL, considerations of in vitro experiments, in vivo
expression patterns of RANK and RANKL, and other potential biologic activities of RANKL are necessary. Expression
analyses and treatment of cells in vitro with RANKL have
shown functional RANK protein expression on the surface
of prostate, breast, osteosarcoma, melanoma, and lung
cancer cell lines. In most studies, RANKL does not seem to
increase proliferation of RANK-expressing tumor cells (46,
47), although increased cell number and protection against
DNA damage-induced cell death, activated by either chemotherapy or g-irradiation, have been observed in certain
cell lines (43). In addition, RANKL will affect a variety of
other tumor cell behaviors potentially relevant to tumor
progression and metastasis, including increased tumor cell
growth in semisolid media, loss of apical–basal polarity,
and stimulated migration and invasion (38, 43, 45, 46, 48,
49). RANKL treatment has been documented to induce a
variety of factors potentially involved in migration, angiogenesis, and invasion, including MMP1, MMP9, the matrix
metalloproteinase inducer EMMPRIN/CD47, ICAM-1, IL-6,
IL-8, and VEGF (47), and it can decrease expression of the
metastasis suppressor serpin 5b/maspin (50).
RANKL-dependent promotion of tumor cell migration
and invasion (as defined in in vitro experiments described
above) may certainly contribute to increased distant metastases observed in vivo. RANKL would be predicted to be
highly expressed in normal (or reactive normal) tissues at
distant sites of metastases, including peripheral lymph
nodes and bone (51), in addition to any RANKL potentially
expressed within the primary tumor. In tumor cell lines,
RANK expression on tumor cells is not strictly required for
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bone metastasis in experimental metastases models. However, 2 in vivo studies are supportive of the skeletal source of
RANKL enhancing metastases of RANK-expressing tumors
directly: (i) the selective prevention of RANK-expressing
tumor cells homing to the bone by RANKL inhibition but
not by bisphosphonates (45) and (ii) the increased skeletal
growth rate of tumor cells with high RANK expression
compared with tumor cell controls with low RANK expression (52). By the same mechanism, RANKL present within
lymph nodes or other metastatic sites could then enhance
metastatic outgrowth of RANK-positive tumors. Consistent
with the above experimental results, a recent analysis
reported that high RANK expression in primary breast
tumors was associated with lymph node involvement and
a higher risk to develop bone metastases (53).
Precise mechanisms for the observed reduction in lung
metastases achieved with RANKL inhibition may depend
upon the model employed. RANKL is not detected within
the primary tumor or inflammatory infiltrate in the spontaneous transgenic MMTV-neu mammary tumor model
(38), which contrasts with the RANKL expression within
tumor-infiltrating T-regs reported after orthotopic transplantation of primary MMTV-neu tumor cells (or a cell line
derived from MMTV-neu tumors; ref. 44). Thus, the clear
reduction in pulmonary metastases by RANKL inhibition
observed in the transgenic MMTV-neu model is not due to
RANKL from infiltrating T lymphocytes; it might instead be
explained by the overall reduced tumor burden observed in
this model (38) and/or other mechanism influencing survival (44) or colonization of metastatic cells. The potential
for a direct contribution of RANKL to tumor progression
and metastases by tumor-infiltrating T lymphocytes
observed in orthotopically transplanted tumors (44) is
intriguing, given the earlier observations that RANKL is
frequently observed (>65%) in the infiltrating monocytic
cells within the stroma of human primary breast tumors
(38) and is upregulated in inflammatory, relative to noninflammatory, human breast cancers (54). Interestingly,
although there is no evidence (either from genetics or
pharmacologic inhibition) that RANKL inhibition is immunosuppressive in vivo (55), RANKL has been shown to
promote the activity of T-regs (56) and macrophages
(57), cell types that are capable of enhancing tumor progression and metastases. Currently, no studies directly
address any function of RANKL on immune components
of the tumor microenvironment, but this hypothesis should
be considered as a potential protumorigenic and prometastatic mechanism along with the direct effects on tumor cells
and/or normal mammary epithelium outlined above.
Clinical-Translational Advances
Targeting RANKL to inhibit tumor-induced osteoclasts
was shown in preclinical proof-of-concept experiments to
be a rational approach for the prevention and treatment of
skeletal complications of malignancy, including metastatic
colonization of the bone. Pharmacodynamic bone resorption biomarkers, including the N-telopeptide of type I
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collagen (NTX), are useful in the translational evaluation of
an osteoclast inhibitor. NTX is a product of bone degradation and a marker of elevated bone resorption, which is
measured as the ratio of urinary NTX to creatinine (uNTX/
Cr). Elevated levels of uNTX/Cr have been associated with
increased risk for experiencing skeletal complications, disease progression, and death in patients with bone metastases (58).
Early versions of RANKL antagonists included recombinant forms of OPG [e.g., Fc-OPG or OPG-Fc (AMGN
0007)]. The first demonstration of biologic activity of
RANKL inhibition in patients with cancer was observed
with patients with multiple myeloma and breast carcinoma
who had radiographic evidence of lytic or mixed bone
disease. OPG-Fc produced rapid dose-related declines in
bone resorption biomarkers (including uNTX/Cr levels;
ref. 59). However, clinical development of OPG forms by
Amgen was terminated because of the relatively short halflife of OPG-Fc in patients with cancer and questions about
the potential safety risk of a neutralizing immune response
against endogenous OPG. Another variant of OPG (CEP37251; Cephalon) was also being developed; however, the
phase I clinical study in healthy postmenopausal women
was apparently terminated (60). A RANKL antibody form
derived from camelidae (i.e., llamas) termed ALX-0141
(Ablynx) has been tested in a phase I study of healthy
postmenopausal women (61). Amgen has developed a fully
human antibody against RANKL (denosumab, AMG 162),
which has shown greater selectivity to human RANKL,
favorable pharmacokinetics, and ease of manufacturing
compared with OPG molecules. Denosumab is a fully
human, immunoglobulin G2 (IgG2) monoclonal antibody, which binds human RANKL with high affinity
(KD ¼ 3 pmol/L) and, as a fully human protein, would not
be predicted to engender an immune response in patients
(62). It binds to soluble and membrane-bound human
RANKL and nonhuman primate RANKL, but not mouse
RANKL. It does not cross-react with other TNF ligand
family members. Similar to OPG, denosumab functions
as a reversible RANKL antagonist, preventing RANKL interaction with RANK and inhibiting osteoclast differentiation, activation, and survival. Denosumab is administered
via a subcutaneous route.
A phase I evaluation in patients with multiple myeloma
or breast cancer metastatic to bone showed a pharmacodynamic effect of denosumab within 24 hours after a single
subcutaneous dose (59). Compared with a single intravenous dose of the antiresorptive bisphosphonate, pamidronate 90 mg, the decrease in bone turnover markers (including uNTX/Cr levels) produced by denosumab was similar in
magnitude but more sustained. In these patients, denosumab was generally well tolerated with no serious adverse
events or antidenosumab antibodies detected.
Phase II studies evaluated the safety and efficacy of
different dosing regimens of denosumab in patients with
cancer, and they informed dose and schedule selection for
subsequent phase III trials. Also, phase II and phase III
studies established the unique mode of action of denosu-
332
Clin Cancer Res; 18(2) January 15, 2012
mab relative to other bone-targeted drugs, specifically nitrogen-containing bisphosphonates (nBP), such as pamidronate and ZA. Bisphosphonates bind to hydroxyapatite bone
mineral surfaces, where they inhibit mature osteoclasts
locally, at sites of bone resorption (63). Intravenous nBPs
have been effective in prevention of skeletal complications
in cancer, but safety and tolerability issues exist, including
renal toxicity and acute-phase reactions. High levels of
uNTX/Cr persist in a substantial proportion of patients
with breast and prostate cancers and other solid tumors
while on intravenous nBP treatment. As a result, these
patients may experience skeletal-related events (including
pathologic fracture, radiation, or surgery to bone, or spinal
cord compression; ref. 58), indicating the need for
improved therapies.
Denosumab reduced bone turnover markers in patients
with cancer in 2 phase II studies. One study in women with
bone metastases from breast cancer concluded that more
patients treated with denosumab had bone turnover suppression compared with intravenous nBP (64). In a second
randomized phase II study, denosumab was tested in
patients with bone metastasis from prostate, breast, or other
solid tumors, or multiple myeloma, who had persistent
elevated levels of uNTx/Cr levels, despite being treated with
intravenous nBP (65). Of these patients, 71% who received
denosumab had reduced levels of uNTx/Cr compared with
29% of patients who continued to receive intravenous nBP.
Furthermore, suppression of serum TRAP5b, an osteoclast
marker, was 2.5-fold greater in patients treated with denosumab compared with patients continuing treatment with
intravenous nBP. In both studies, the safety profile of
denosumab was consistent with a cancer population receiving systemic antineoplastic treatment.
Evaluation of the phase II results, using a population
pharmacokinetic–pharmacodynamic model, suggested
that a dose of 120 mg denosumab given every 4 weeks
would suppress uNTX/Cr levels by more than 90% in most
patients (64), and pharmacokinetic analyses of serum
denosumab levels indicated a mean half-life of approximately 30 days (66). Altogether, these phase II studies
clearly defined the efficacy of denosumab in patients with
cancer, independent of the tumor type, and established
the ability of denosumab to control hyperactive bone
resorption.
Skeletal-related events are serious irreversible complications of tumors that metastasize to the bone. To evaluate the
ability of denosumab to prevent skeletal-related events, 3
large phase III clinical studies were done in patients who
had bone metastases from breast cancer (67), castrationresistant prostate cancer (CRPC; ref. 68), and any other
advanced cancer (excluding breast and prostate) or multiple
myeloma (69). Each trial was a randomized, double-blind,
double-dummy, active controlled comparison of s.c. denosumab 120 mg with i.v. ZA 4 mg (adjusted for creatinine
clearance) every 4 weeks. The primary endpoint for each
study was time to first on-study skeletal-related event. In the
breast cancer trial (n ¼ 2,046), denosumab significantly
delayed the time to first skeletal-related event by 18% versus
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RANKL in Tumorigenesis and Metastasis
ZA (P ¼ 0.01, superiority) and reduced the risk of multiple
skeletal-related events by 23% versus ZA (P ¼ 0.001, superiority), showing the durability of the positive effect on
skeletal complications (67). In men with CRPC and bone
metastases (n ¼ 1,904), denosumab significantly delayed
the time to first on-study skeletal-related event by 18%
compared with ZA (P ¼ 0.008, superiority) and the time
to first and subsequent skeletal-related event (P ¼ 0.008,
superiority; ref. 68). Finally, Henry and colleagues (69)
reported that denosumab delayed time to first on-study
skeletal-related event (HR 0.84; P ¼ 0.0007, noninferiority)
compared with ZA in patients with bone metastases from a
variety of solid tumors (excluding prostate and breast cancer, but representing more than 50 different tumor types) or
multiple myeloma. For all 3 studies, time to disease progression and overall survival rates were similar between the
denosumab- and ZA-treated cohorts. The overall rate of onstudy adverse events was also similar between the 2 cohorts,
including infrequent observations of osteonecrosis of the
jaw typically associated with previously reported risk factors. Rates of adverse events potentially associated with
renal toxicity and acute-phase reactions were elevated in
patients treated with ZA, whereas a greater incidence of
hypocalcemia was observed in the denosumab cohorts.
Hypocalcemia was manageable, was not associated with
significant clinical sequelae, and was consistent with denosumab’s mechanism of action. These 3 identically designed
pivotal clinical studies were the basis upon which denosumab achieved approval by the U.S. Food and Drug Administration in November 2010 for prevention of skeletalrelated events in patients with bone metastases from solid
tumors.
Conclusions
The early findings of an essential role of RANKL in
physiologic osteoclastogenesis was the fundamental
rationale for targeting this pathway as a treatment for
skeletal complications in cancer, and a therapeutic antibody, denosumab, has been successfully developed in the
clinic for this application in patients with solid tumor
bone metastases. As preclinical experiments have shown,
the interference with the bone microenvironment and
potent reduction in bone resorption with a RANKL inhibitor also has potential utility in the inhibition of early
metastatic colonization. Consistent with this hypothesis,
denosumab was recently shown to be effective in delaying
development of bone metastasis in men with CRPC (70),
and this study represents the first large randomized study
to show that targeting the bone microenvironment has
this effect in patients with cancer. This hypothesis is also
currently being addressed in women with early-stage
breast cancer at high risk of disease recurrence to determine whether denosumab in combination with standardof-care adjuvant and/or neoadjuvant cancer treatment
will improve bone metastasis–free and disease-free survival compared with standard-of-care treatment alone
(71).
www.aacrjournals.org
Although more recent preclinical basic research in
rodents has indicated that RANKL can promote a spectrum
of effects relevant to cancer initiation, progression, and
metastasis via direct effects on either tumor cells or the
mammary epithelium, significant gaps remain in the translation of these more recent findings to the clinic. The
prominent role of RANKL as a paracrine effector of progesterone action in the mouse mammary epithelium clearly
has implications in humans, given the potential role for
progesterone specifically as a risk factor in human breast
cancer, as supported by its well-established mitogenic
effects in the breast (72) and by large epidemiologic studies
(73). However, fundamental differences exist in the anatomy and hormone responsiveness of the rodent mammary
gland compared with the primate, which may limit the
utility of the preclinical findings. Data linking the RANKL
pathway (RANKL, RANK, and OPG) expression with hormone exposure and breast density and proliferation in
humans, or in a suitable nonhuman primate model, are
needed to show relevance of the progesterone and RANKL
pathway association. To address any association of the
RANK/RANKL pathway with cancer risk or progression,
genome-wide association studies or analysis of mRNA or
protein expression may be helpful, but each approach has
its limitations. Using a candidate gene approach, a recent
genetic association study has associated a single nucleotide
polymorphism in the RANK gene with breast cancer risk
(74). In addition to progesterone, multiple other potential
stimuli of RANKL in cancer may exist (e.g., PTHrP, prolactin, tumor stromal cells, infiltrating T lymphocytes), and
further studies are necessary not only to verify the many
diverse sources of RANKL with well-validated methodologies but also to determine whether any of these RANKL
sources are associated with disease outcome. Likewise, it
will be crucial to define any tumor subpopulations that
express RANK, again using well-validated and specific
approaches, and to relate RANK expression with additional disease outcomes. Recent analysis of RANK mRNA
expression in human breast cancer biopsies indicates
relatively increased expression in the basal tumor subtype
and an association of higher RANK mRNA expression
with poor survival (53). Finally, the identification of
tumor-specific (or mammary epithelial-specific) biomarkers of a RANKL response would aid in the translational
evaluation of this drug in extraskeletal tissues and, ultimately, the testing of the current preclinical hypotheses in
patients.
Disclosure of Potential Conflicts of Interest
W. C. Dougall: employment and stockholder, Amgen Inc.
Acknowledgments
I would like to acknowledge the editorial assistance of Albert Rhee and
Geoff Smith. I would also like to thank Michelle Blake, Dan Branstetter,
Allison Jacob, and Lanny Kirsch for their critique of this article.
Received August 16, 2011; revised October 5, 2011; accepted October 6,
2011; published OnlineFirst October 26, 2011.
Clin Cancer Res; 18(2) January 15, 2012
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333
Published OnlineFirst October 26, 2011; DOI: 10.1158/1078-0432.CCR-10-2507
Dougall
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Published OnlineFirst October 26, 2011; DOI: 10.1158/1078-0432.CCR-10-2507
Molecular Pathways: Osteoclast-Dependent and
Osteoclast-Independent Roles of the RANKL/RANK/OPG Pathway
in Tumorigenesis and Metastasis
William C. Dougall
Clin Cancer Res 2012;18:326-335. Published OnlineFirst October 26, 2011.
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