Molecular Pathways: Osteoclast-Dependent and Osteoclast ...bitors, such as OPG and RANK-Fc, have...

Molecular Pathways

Molecular Pathways: Osteoclast-Dependent andOsteoclast-Independent Roles of the RANKL/RANK/OPGPathway in Tumorigenesis and Metastasis

William C. Dougall

AbstractReceptor 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 ofRANKL toOPG is upregulated,which leads to increasedosteoclast-mediatedbonedestruction. 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 anti-

RANKL 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 distinctmechanisms, 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 theRANK/RANKL pathway

The cells that control bone remodeling include the oste-oblast, which deposits new bone, and the osteoclast, aspecialized cell of hematopoietic origin that resorbs theinorganic and organic matrix of the bone. Osteoblast andosteoclast function are coordinately regulated in normalbone remodeling. Identification of the receptor activator ofnuclear factor-kappa B (RANK), RANK ligand (RANKL),and osteoprotegerin (OPG) pathway in the mid-1990srevealed a key molecular axis for osteoclast formation,function, and survival, and it provided crucial insights intothe regulation of normal, physiologic bone remodeling.Functional genomics in mice showed that RANKL

(TNFSF11), a member of the TNF ligand superfamily, andRANK (TNFRSF11a), the cognate TNFR family receptor forRANKL, were essential for osteoclastogenesis in vivo. Afunctional interaction between RANKL, expressed by bonestromal cells of the osteoblast lineage, and RANK, expressedby osteoclast precursors of hematopoietic myeloid lineage,is necessary for osteoclast differentiation, survival, andactivation. Knockout mice lacking either RANK or RANKLdevelop significant osteopetrosis resulting from a lack ofosteoclasts and absence of bone resorption (1, 2). OPG(TNFRSF11b), another member of the TNFR superfamily,can also bind RANKL and functions as a soluble decoyreceptor for RANKL. The critical role of OPG in osteoclas-togenesis and bone remodeling was first shown by theincreased bone mass as a result of reduced osteoclast num-bers observed in transgenic mice overexpressing OPG (3)and, conversely, the osteopenia observed in OPG knockoutmice (4). Given that the precise and balanced interaction ofRANK, RANKL, and OPG is critical for osteoclastogenesisand, therefore, themaintenance of homeostatic bone remo-deling and bone mass, it was hypothesized that this path-way may become dysregulated within the bone duringdisease states and contribute to pathologic bone loss, suchas postmenopausal osteoporosis or cancer-induced bonedestruction (Fig. 1).

Author's Affiliation: Department of Hematology and Oncology Research,Amgen Inc., Seattle, Washington

Corresponding Author: William C. Dougall, Department of Hemato-logy 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.

ClinicalCancer

Research

Clin Cancer Res; 18(2) January 15, 2012326

on February 17, 2021. © 2012 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 26, 2011; DOI: 10.1158/1078-0432.CCR-10-2507

http://clincancerres.aacrjournals.org/

A unique feature of skeletal metastases and multiplemyeloma is the active involvement of the host, particu-larly osteoclasts, in the pathophysiologic process leadingto bone destruction and skeletal tumor growth. Acceler-ated osteoclast action and subsequent increased boneresorption have been operatively linked to a full spectrumof skeletal complications (e.g., hypercalcemia, pathologicfracture, bone pain, spinal cord compression, surgery orradiation treatment to the bone, etc.) that result frombone metastases and multiple myeloma. Osteoclastinvolvement is observed in all types of cancer-inducedbone destruction, regardless of the lesions’ radiographicappearance as either osteolytic or osteoblastic, and irre-spective of the tumor type of origin (5–7).In addition to causing destructive bone loss and associ-

ated clinical sequelae, tumor-induced osteoclasts contrib-ute to the establishment, growth, and survival of tumors.

Products of osteoclastic bone resorption, including matrix-derived growth factors [e.g., TGF-b, insulin-like growthfactor I (IGF-I)] and elevated calcium levels, increase tumorcell proliferation and survival and induce further produc-tion of osteolytic and osteoblastic factors, thereby creating apositive feedback loop known as the "vicious cycle" (7). Asosteoclasts are highly related to macrophages, it is perhapsnot surprising that many protumorigenic factors can beproduced 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 additionto the production of matrix metalloproteinases (e.g.,MMP-7, MMP-9, MMP-14; refs. 8, 9). Thus, the osteoclastmay indirectly provide factors that contribute to skeletaltumor establishment and progression, as a consequence ofbone matrix turnover, in addition to cellular production,

© 2011 American Association for Cancer Research

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Figure 1. Bone-intrinsic functionality of the RANKL pathway in normal and pathologic osteoclastogenesis. A, normal bone turnover and/orremodeling observed in healthy physiologic systems. Bone stromal cells, including cells of the osteoblast lineage, provide a limited amount of RANKL, whichleads to osteoclast differentiation, survival, and activation and subsequent bone resorption. Resorption is balanced by osteoblast-dependent new boneformation. If occult micrometastases were to exist, they would remain dormant because of the low level of bone turnover (37). B, tumor-induced highbone 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, andactivity, which increases the rate of bone remodeling and/or turnover. (4) Pathologic bone remodeling, characterized by increased osteoclast and osteoblastactivities, causes a spectrum of bone lesions in patients with bonemetastasis, ranging from predominately osteoblastic to predominately osteolytic. Multiplemyeloma gives rise to purely lytic bone lesions. (5) As a result of the increased osteoclast function, many changes occur within the reactive bonemicroenvironment. These changes include increases in local levels of calcium, release of activated growth factors from the bone matrix, and increasedproduction of growth and/or angiogenic factors by the osteoclast. (6) Increased tumor cell growth and survival, including the outgrowth of dormantmicrometastases, occur as a result of these bone matrix–and osteoclast-derived factors. (7) Skeletal tumor cells respond to these bone signals with furtherproduction of additional proresorptive factors, generating a feed-forward loop known as the "vicious cycle."

RANKL in Tumorigenesis and Metastasis

www.aacrjournals.org Clin Cancer Res; 18(2) January 15, 2012 327




independent of bone resorption. Initial studies of breastcancer by Stephen Paget in 1889 identified the bone as anoptimal site (or "soil") for distant metastases ("seed";ref. 10). It is now clear that many other tumors, includingprostate, lung, renal, melanoma, and thyroid, have a highpropensity tometastasize to bone, highlighting the skeletonas a fecundmetastatic site capable of harboring early tumorcolonization and actively promoting the growth of skeletaltumors.

The RANKL, RANK, OPG pathway in bone- andcancer-induced bone disease

In each of these aforementioned pathologic processes(bone destruction, metastatic establishment, and tumorprogression), the tumor has coopted the host by upsettingthe balance of 2 key factors that normally govern physio-logic osteoclast formation and bone remodeling: RANKLand OPG (Fig. 1). The cancer-induced signals capable ofaltering the normally well-balanced RANKL-to-OPG ratiocan be extremely diverse, reflecting the different sources ofRANKL and OPG, the assortment of tumor types that affectbone, and the heterogeneity of individual tumor types(7, 11). More importantly, these diverse signals andmechanisms that elevate osteoclastogenesis and bonedestruction converge on the RANKL pathway. Many differ-ent cytokines or factors produced by skeletal tumors [e.g.,IL-1b, IL-6, IL-8, IL-11, IL-17, macrophage inflammatoryprotein 1a, TNFa, parathyroid hormone-releasing protein(PTHrP), prostaglandin E (PGE2)] cause increased RANKLproduction by stromal cells in the bonemicroenvironment,including cells of the osteoblast lineage. OPG, the normaldecoy receptor for RANKL, can also be downregulated bytumors via variousmechanisms; reduced synthesis or activedegradation of OPG in the bone is observed as a conse-quence of tumor-derived factors (11). Certain factors inmetastatic cancer have dual effects on the RANKL-to-OPGratio. For instance, PTHrP, IL-1, and PGE2 have been shownto act on the bone stroma and stimulate osteoclast activityby increasing RANKL and decreasing OPG simultaneously(7). T lymphocytes, including activated T cells (12) andCD4þCD25þ T-regulatory cells (T-reg; ref. 13), may beanother source of RANKL in bone metastases or otherextraskeletal cancer settings in which RANKL may function(see below). Multiple myeloma cells can increase RANKLproduction in T lymphocytes (14); however, functionalevidence for T-cell contribution of RANKL in cancer–boneinteractions is lacking, largely because most preclinicalbone cancer models are studied in immunocompromisedhosts.

Increases in RANKL that lead to cancer-induced osteo-clastogenesis are not limited to infiltrating immune cells orreactive changes in thebone stroma causedby the tumorbutcan be contributed by the tumor cells themselves. RANKLexpression has also been reported on some tumor types,including breast cancer, prostate cancer,multiplemyeloma,and renal carcinoma (as described below). RANKL pro-duced by tumor cells can increase osteoclastogenesis in vitro(15), suggesting that the tumor cell localized in the skeleton

may also directly contribute an osteoclastogenic signal. It iscertainly possible that the bone microenvironment pro-vides signals that can increase RANKL on metastatic tumorcells, which in turn enhances further osteoclast activity andcreates a feed-forward loop. For instance, RANKL increaseshave been observed in prostate tumor cell lines after treat-ment with TGF-b (16), a product of bone resorption, orupon stimulation of the epithelial–mesenchymal transition(EMT) by TGF-b plus epidermal growth factor treatment ortransfection with SNAIL (17). Intriguingly, EMT changes intumor cells residing in the bone have been linked to amoreinvasive phenotype, and development of overt bonemetas-tases from dormant micrometastases (17, 18), although afunctional role for RANKL in this transition, has not yetbeen defined. Consistent with the hypothesis that RANKLproduction by tumor cells accelerates osteoclast formationand bone metastasis is the observation that a high expres-sion level of RANKL in primary renal cell carcinoma isassociated with a shorter bone metastasis–free survival(19). A similar relationship between high RANKL expres-sion levels in primary tumors and development of bonemetastases has also been described for patients with hepa-tocellular carcinoma (20).

Currently, no definitive data correlate circulating levels ofRANKL in serum with bone metastases. This lack of datamay reflect the preponderance of the membrane-boundversus soluble form of RANKL, the relatively low levels ofRANKLdetected in the serum, or the technical limitations ofsuch assays (21). It is also likely that increases in RANKLremain localized to the bone lesion, where focal activationof bone remodeling and increased osteoclast activity areclosely juxtaposed to tumor infiltration (22). In contrast, arelationship between the higher ratio of serum RANKL toOPG and a greater extent of bone lesions has been observedin multiple myeloma (23), which may reflect either theextensive tumor cell production of RANKL or the systemicnature of this hematologic tumor.

To address the functional contribution of RANKL tocancer-induced bone disease, pharmacologic RANKL inhi-bitors, such as OPG and RANK-Fc, have been tested inrodent models, and these studies have been the subject ofrecent reviews (11). Preclinical models of cancer bonemetastasis and multiple myeloma typically develop osteo-lytic, osteoblastic, or mixed lesions after systemic or intra-tibial injection in rodents. RANKL inhibition has beentested in models representing many different tumor types,includingmultiplemyeloma, breast cancer, prostate cancer,lung cancer, colon cancer, etc., and in each case has beenshown to effectively reduce tumor-induced bone lesions(11). Given the specific mechanism of RANKL inhibition,the observed broad activity across lesion types suggests thatosteoclastic activity may be a requisite element for bothosteolytic and osteoblastic lesions. In addition to the ben-eficial effect on bone lesions, RANKL blockade has alsoreduced other tumor-associated sequelae, including bonepain (24) and hypercalcemia of malignancy (25).

On the basis of the observed reciprocal feedback (thevicious cycle) occurring between bone and tumor, one

Dougall

Clin Cancer Res; 18(2) January 15, 2012 Clinical Cancer Research328




would predict that reduction of bone resorption would notonly reduce the tumor-induced bone destruction but alsoslow skeletal tumor progression. Studies using preclinicalcancer–bone models representing different solid tumorsand multiple myeloma have shown a substantial reductionin tumor growth in the bone as a consequence of the potentosteoclast blockade achieved with RANKL inhibition (6,7, 26). The reductions in skeletal tumor burden uponRANKL inhibition are dependent on the bone microenvi-ronment and are associated with increased tumor cellapoptosis, decreased tumor cell proliferation, and increasedsurvival of tumor-bearing mice (27–30). RANKL inhibitioneffectively reduces skeletal tumor growth as amonotherapy,but as would be predicted by an approach that targets thebone microenvironment and reduces local growth factorand calcium production, the reduction in skeletal tumorburden 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 bonemicroenvironment actively contributes to the processes ofearly tumor colonization and metastatic outgrowth, thenskeletal metastases might be prevented or delayed throughpharmacologic blockade of bone turnover (34), withRANKL inhibition as a potential approach. To test thishypothesis in preclinical models, however, one must con-sider limitations of experimental bone metastases models,including the observation that skeletal growth of xeno-grafted human tumor cells may ultimately achieve a criticalmass capable of self-maintenance independent of the bonemicroenvironment. To address this limitation, it has beennecessary to model tumor–bone interactions at early stagesof bone metastasis and colonization using high-sensitivity,small-animal imaging techniques. Prophylactic treatmentofmice with the RANKL inhibitor OPG-Fc was employed toreduce baseline osteoclast activity and bone resorptionprior to inoculation of breast tumor MDA-231 cells. Thisstrategy subsequently delayed de novo formation of meta-static skeletal tumors as monitored by bioluminescentimaging, presumably by arresting the vicious cycle support-ing initial tumor growth (27).

The RANKL pathway in normal mammary biology andcancerIn addition to severe osteopetrosis, RANK and RANKL

knockout mice manifested a lactation defect, revealing anintrinsic functionality of the pathway in mammary epithe-lium that is also relevant to mammary tumorigenesis andmetastases. The failure of RANK or RANKL knockout miceto lactate was due to a marked inability to develop lobulo-alveolar mammary structures, which normally undergo amassive expansion under hormonal control during preg-nancy and eventually differentiate into milk-secreting tis-sue. Proliferation and survival of the mammary epitheliumare reduced in the absence of a productive RANKL/RANKsignal, and transplantation experiments showed this defectto be autonomous to mammary epithelial tissue (35).RANKL expression is greatly increased in luminalmammary

epithelial cells at midgestation pregnancy and can beinduced by factors such as prolactin, PTHrP, and proges-terone (35). In mice, RANK expression is observed at lowlevels in luminal and basal cells of the mammary epithe-lium and becomes more highly expressed at midgestationpregnancy, most selectively at ductal branch points (36).

Genetic studies revealed that both RANKL and proges-terone receptor (PR) function at similar stages in lactationalmorphogenesis and that these proteins are colocalized inluminalmammary epithelial cells. Recent experiments haveshown that the major mitogenic effect of progesteroneoccurs via increases inRANKLwithinPRþ luminal epithelialcells. RANKL, acting as a paracrine factor, then inducesmitogenesis of neighboring estrogen receptor (ER)�/PR�

mammary epithelial cells (37–39). Moreover, other recentstudies have shown that RANKL can mediate the nonpro-liferative expansion of the mouse mammary gland viaincreases in the number and regenerative capacity of mam-mary stem cells (MaSC). Despite their ER�/PR� phenotype,MaSC are profoundly responsive to ovarian hormone sig-naling, and RANKL was identified as a paracrine effector ofthe progesterone-dependent effects on MaSC occurringduring either gestation or estrous cycles (40, 41).

RANKL promotes mammary tumorigenesisThe fundamental roles of the RANKL pathway in the

normal physiology of the mammary gland have significantimplications in cancer and tumorigenesis. RANKL-drivenhormone (progesterone)-dependent proliferation, survival,and nonproliferative expansion of MaSC could each con-tribute to mammary cancer initiation, progression, andrecurrence (Fig. 2). During normal lactational morphogen-esis, it is well characterized that RANKandRANKL adhere tostrict, yet overlapping, spatial–temporal expression patternswithin the mammary epithelium under control of lactationhormones. However, when RANKL or RANK is overex-pressed in the mammary epithelium [via mouse transgenicmodels using the mouse mammary tumor virus (MMTV)promoter] in the absence of strict hormonal control, inap-propriate mammary proliferation is observed (36, 42).Despite the aberrant proliferation (including hyperplasia)induced by an overactive RANKL pathway in these 2 mod-els, spontaneous mammary tumors were not observed inaged virgin mice. However, accelerated preneoplasias andincreased mammary tumor formation were observed inMMTV-RANK mice after multiparity or treatment withthe carcinogen 7,12-dimethylbenz(a)anthracene (DMBA)in combination with a synthetic progestin hormone[medroxyprogesterone acetate (MPA); ref. 38], settings inwhich mammary epithelial RANKL expression is increasedowing to increased sex hormone levels. Pharmacologicblockade of RANKL decreased incidence and delayed onsetof mammary tumors induced by DMBA and MPA in bothMMTV-RANK transgenicmice andwild-typemice (38). Thereduction inmammary tumor formation was preceded by areduction in preneoplasias and correlated with rapid andsustained reductions in hormone-induced mammary epi-thelial proliferation and cyclin D1 expression, as well as






increased apoptosis, suggesting that RANKL affects earlystages of tumor formation. A similar reduction inmammarytumor formation (also induced byMPA and/or DMBA)wasobserved by Schramek and colleagues (43) in mice, inwhich RANK had been selectively deleted from the mam-mary epithelium by a tissue-specific Cre recombinase–mediated approach. This genetic approach also revealedthat the RANKL pathway protects against DNA damage-induced cell death and expands the putative stem cell

component of mammary tumors. In a model of mammarycancer induced by the c-neu oncogene in the absence ofexogenous hormone (MMTV-neu transgenic mice), inhibi-tion of RANKL (beginning at 5months of age) did not affectmedian time to spontaneous mammary tumor formation,but it did decrease the number of preneoplastic lesions andmammary tumors (38). Mammary tumor formation wasnot reduced with another inhibitor of bone resorption [i.e.,the nitrogen-containing bisphosphonate zoledronic acid

© 2011 American Association for Cancer Research

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Figure 2. Direct protumorigenic and prometastatic activities of RANKL. Using mammary transformation as a model, several different sources of RANKL areindicated, which may impact multiple steps in tumor progression. For instance, hormonal influences (e.g., progesterone, PTHrP, prolactin) may increaseRANKL not only in the normal mammary epithelium but also in preneoplastic, tumor, and metastatic lesions. RANKL has also been observed withinpreneoplastic andneoplastic cells independent of hormone influences.RANKL expressedonT lymphocytesmaypotentially be important atmultiple stages incancer, from preneoplasias through metastases. Certain metastatic sites, such as the lymph node and bone, are rich sources of RANKL. Different RANKL-dependent 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 enhancednumbers and regenerative potential of MaSC or the putative stem cell component of tumors. Other RANKL-dependent activities on tumor cells potentiallyrelevant to distantmetastatic spread are reflected by the in vitro observations of enhancedmigration and invasion or increased growth in a semisolidmedium.

Dougall





(ZA); ref. 38]. In line with the observed reductions inmammary epithelial biomarkers upon RANKL inhibitionand the protection against tumors observed in the mam-mary-selective knockout of RANK, these data confirm directosteoclast-independent functionality of the RANKL path-way in cancer.

A direct role of the RANKL pathway in distantmetastasesThere is also preclinical evidence that blockade of RANKL

will reduce distant metastasis, potentially via mechanismsdistinct from thebone-intrinsic antiosteoclast effect (Fig. 2).Treatment of transgenic MMTV-neu mice with RANK-Fcsignificantly decreased spontaneous lung metastases (38).Consistent with this observation, crossing the MMTV-neumice with RANK heterozygotes reduced the incidence ofspontaneous lung metastases (44). Reductions in bonemetastases or lung metastases have been achieved withpharmacologic blockade of RANKL in animals bearingRANK-positivemelanoma cells (45) or transplanted prima-ryMMTV-neu tumor cells (44). Reciprocally, enhanced lungmetastases have been observed upon systemic RANKL expo-sure in mice bearing orthotopically transplanted tumorcells, originating from either a RANK-positive humanbreasttumor cell line or tumor cells derived fromMMTV-neumice(44).To gain mechanistic insight into the prometastatic activ-

ity of RANKL, considerations of in vitro experiments, in vivoexpression patterns of RANK and RANKL, and other poten-tial biologic activities of RANKL are necessary. Expressionanalyses and treatment of cells in vitro with RANKL haveshown functional RANK protein expression on the surfaceof prostate, breast, osteosarcoma, melanoma, and lungcancer cell lines. In most studies, RANKL does not seem toincrease proliferation of RANK-expressing tumor cells (46,47), although increased cell number and protection againstDNA damage-induced cell death, activated by either che-motherapy or g-irradiation, have been observed in certaincell lines (43). In addition, RANKL will affect a variety ofother tumor cell behaviors potentially relevant to tumorprogression and metastasis, including increased tumor cellgrowth 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 avariety of factors potentially involved in migration, angio-genesis, and invasion, including MMP1, MMP9, the matrixmetalloproteinase inducer EMMPRIN/CD47, ICAM-1, IL-6,IL-8, and VEGF (47), and it can decrease expression of themetastasis suppressor serpin 5b/maspin (50).RANKL-dependent promotion of tumor cell migration

and invasion (as defined in in vitro experiments describedabove) may certainly contribute to increased distant metas-tases observed in vivo. RANKL would be predicted to behighly expressed in normal (or reactive normal) tissues atdistant sites of metastases, including peripheral lymphnodes and bone (51), in addition to any RANKL potentiallyexpressed within the primary tumor. In tumor cell lines,RANK expression on tumor cells is not strictly required for

bone metastasis in experimental metastases models. How-ever, 2 in vivo studies are supportive of the skeletal source ofRANKL enhancing metastases of RANK-expressing tumorsdirectly: (i) the selective prevention of RANK-expressingtumor cells homing to the bone by RANKL inhibition butnot by bisphosphonates (45) and (ii) the increased skeletalgrowth rate of tumor cells with high RANK expressioncompared with tumor cell controls with low RANK expres-sion (52). By the same mechanism, RANKL present withinlymph nodes or other metastatic sites could then enhancemetastatic outgrowth of RANK-positive tumors. Consistentwith the above experimental results, a recent analysisreported that high RANK expression in primary breasttumors was associated with lymph node involvement anda higher risk to develop bone metastases (53).

Precise mechanisms for the observed reduction in lungmetastases achieved with RANKL inhibition may dependupon the model employed. RANKL is not detected withinthe primary tumor or inflammatory infiltrate in the spon-taneous transgenic MMTV-neu mammary tumor model(38), which contrasts with the RANKL expression withintumor-infiltrating T-regs reported after orthotopic trans-plantation of primary MMTV-neu tumor cells (or a cell linederived from MMTV-neu tumors; ref. 44). Thus, the clearreduction in pulmonary metastases by RANKL inhibitionobserved in the transgenic MMTV-neu model is not due toRANKL from infiltrating T lymphocytes; it might instead beexplained by the overall reduced tumor burden observed inthis model (38) and/or other mechanism influencing sur-vival (44) or colonization of metastatic cells. The potentialfor a direct contribution of RANKL to tumor progressionand metastases by tumor-infiltrating T lymphocytesobserved in orthotopically transplanted tumors (44) isintriguing, given the earlier observations that RANKL isfrequently observed (>65%) in the infiltrating monocyticcells within the stroma of human primary breast tumors(38) and is upregulated in inflammatory, relative to non-inflammatory, human breast cancers (54). Interestingly,although there is no evidence (either from genetics orpharmacologic inhibition) that RANKL inhibition is immu-nosuppressive in vivo (55), RANKL has been shown topromote the activity of T-regs (56) and macrophages(57), cell types that are capable of enhancing tumor pro-gression and metastases. Currently, no studies directlyaddress any function of RANKL on immune componentsof the tumormicroenvironment, but this hypothesis shouldbe considered as a potential protumorigenic and prometa-staticmechanismalongwith thedirect effects on tumor cellsand/or normal mammary epithelium outlined above.

Clinical-Translational Advances

Targeting RANKL to inhibit tumor-induced osteoclastswas shown in preclinical proof-of-concept experiments tobe a rational approach for the prevention and treatment ofskeletal complications of malignancy, including metastaticcolonization of the bone. Pharmacodynamic bone resorp-tion biomarkers, including the N-telopeptide of type I






collagen (NTX), are useful in the translational evaluation ofan osteoclast inhibitor. NTX is a product of bone degrada-tion and a marker of elevated bone resorption, which ismeasured as the ratio of urinary NTX to creatinine (uNTX/Cr). Elevated levels of uNTX/Cr have been associated withincreased risk for experiencing skeletal complications, dis-ease progression, and death in patients with bone metas-tases (58).

Early versions of RANKL antagonists included recombi-nant forms of OPG [e.g., Fc-OPG or OPG-Fc (AMGN0007)]. The first demonstration of biologic activity ofRANKL inhibition in patients with cancer was observedwith patients withmultiple myeloma and breast carcinomawho had radiographic evidence of lytic or mixed bonedisease. OPG-Fc produced rapid dose-related declines inbone resorption biomarkers (including uNTX/Cr levels;ref. 59). However, clinical development of OPG forms byAmgen was terminated because of the relatively short half-life of OPG-Fc in patients with cancer and questions aboutthe potential safety risk of a neutralizing immune responseagainst endogenous OPG. Another variant of OPG (CEP-37251; Cephalon) was also being developed; however, thephase I clinical study in healthy postmenopausal womenwas apparently terminated (60). A RANKL antibody formderived from camelidae (i.e., llamas) termed ALX-0141(Ablynx) has been tested in a phase I study of healthypostmenopausal women (61). Amgenhas developed a fullyhuman antibody against RANKL (denosumab, AMG 162),which has shown greater selectivity to human RANKL,favorable pharmacokinetics, and ease of manufacturingcompared with OPG molecules. Denosumab is a fullyhuman, immunoglobulin G2 (IgG2) monoclonal anti-body, which binds human RANKL with high affinity(KD ¼ 3 pmol/L) and, as a fully human protein, would notbe predicted to engender an immune response in patients(62). It binds to soluble and membrane-bound humanRANKL and nonhuman primate RANKL, but not mouseRANKL. It does not cross-react with other TNF ligandfamily members. Similar to OPG, denosumab functionsas a reversible RANKL antagonist, preventing RANKL inter-action with RANK and inhibiting osteoclast differentia-tion, activation, and survival. Denosumab is administeredvia a subcutaneous route.

A phase I evaluation in patients with multiple myelomaor breast cancer metastatic to bone showed a pharmacody-namic effect of denosumab within 24 hours after a singlesubcutaneous dose (59). Compared with a single intrave-nous dose of the antiresorptive bisphosphonate, pamidro-nate 90 mg, the decrease in bone turnover markers (includ-ing uNTX/Cr levels) produced by denosumabwas similar inmagnitude but more sustained. In these patients, denosu-mab was generally well tolerated with no serious adverseevents or antidenosumab antibodies detected.

Phase II studies evaluated the safety and efficacy ofdifferent dosing regimens of denosumab in patients withcancer, and they informed dose and schedule selection forsubsequent phase III trials. Also, phase II and phase IIIstudies established the unique mode of action of denosu-

mab relative to other bone-targeted drugs, specifically nitro-gen-containing bisphosphonates (nBP), such as pamidro-nate and ZA. Bisphosphonates bind to hydroxyapatite bonemineral surfaces, where they inhibit mature osteoclastslocally, at sites of bone resorption (63). Intravenous nBPshave been effective in prevention of skeletal complicationsin cancer, but safety and tolerability issues exist, includingrenal toxicity and acute-phase reactions. High levels ofuNTX/Cr persist in a substantial proportion of patientswith breast and prostate cancers and other solid tumorswhile on intravenous nBP treatment. As a result, thesepatients may experience skeletal-related events (includingpathologic fracture, radiation, or surgery to bone, or spinalcord compression; ref. 58), indicating the need forimproved therapies.

Denosumab reduced bone turnover markers in patientswith cancer in 2 phase II studies. One study in women withbone metastases from breast cancer concluded that morepatients treated with denosumab had bone turnover sup-pression compared with intravenous nBP (64). In a secondrandomized phase II study, denosumab was tested inpatients with bonemetastasis fromprostate, breast, or othersolid tumors, or multiple myeloma, who had persistentelevated levels of uNTx/Cr levels, despite being treated withintravenous nBP (65). Of these patients, 71%who receiveddenosumab had reduced levels of uNTx/Cr compared with29% of patients who continued to receive intravenous nBP.Furthermore, suppression of serum TRAP5b, an osteoclastmarker, was 2.5-fold greater in patients treated with deno-sumab compared with patients continuing treatment withintravenous nBP. In both studies, the safety profile ofdenosumabwas consistent with a cancer population receiv-ing systemic antineoplastic treatment.

Evaluation of the phase II results, using a populationpharmacokinetic–pharmacodynamic model, suggestedthat a dose of 120 mg denosumab given every 4 weekswould suppress uNTX/Cr levels by more than 90% in mostpatients (64), and pharmacokinetic analyses of serumdenosumab levels indicated a mean half-life of approxi-mately 30 days (66). Altogether, these phase II studiesclearly defined the efficacy of denosumab in patients withcancer, independent of the tumor type, and establishedthe ability of denosumab to control hyperactive boneresorption.

Skeletal-related events are serious irreversible complica-tions of tumors thatmetastasize to the bone. To evaluate theability of denosumab to prevent skeletal-related events, 3large phase III clinical studies were done in patients whohad bone metastases from breast cancer (67), castration-resistant prostate cancer (CRPC; ref. 68), and any otheradvanced cancer (excluding breast andprostate) ormultiplemyeloma (69). Each trial was a randomized, double-blind,double-dummy, active controlled comparison of s.c. deno-sumab 120 mg with i.v. ZA 4 mg (adjusted for creatinineclearance) every 4 weeks. The primary endpoint for eachstudy was time to first on-study skeletal-related event. In thebreast cancer trial (n ¼ 2,046), denosumab significantlydelayed the time to first skeletal-related event by 18%versus

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ZA (P ¼ 0.01, superiority) and reduced the risk of multipleskeletal-related events by 23% versus ZA (P ¼ 0.001, supe-riority), showing the durability of the positive effect onskeletal complications (67). In men with CRPC and bonemetastases (n ¼ 1,904), denosumab significantly delayedthe time to first on-study skeletal-related event by 18%compared with ZA (P ¼ 0.008, superiority) and the timeto 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-studyskeletal-related event (HR 0.84; P¼ 0.0007, noninferiority)compared with ZA in patients with bone metastases from avariety of solid tumors (excluding prostate and breast can-cer, but representingmore than 50 different tumor types) ormultiple myeloma. For all 3 studies, time to disease pro-gression and overall survival rates were similar between thedenosumab- and ZA-treated cohorts. The overall rate of on-study adverse events was also similar between the 2 cohorts,including infrequent observations of osteonecrosis of thejaw typically associated with previously reported risk fac-tors. Rates of adverse events potentially associated withrenal toxicity and acute-phase reactions were elevated inpatients treated with ZA, whereas a greater incidence ofhypocalcemia was observed in the denosumab cohorts.Hypocalcemia was manageable, was not associated withsignificant clinical sequelae, and was consistent with deno-sumab’s mechanism of action. These 3 identically designedpivotal clinical studies were the basis upon which denosu-mab achieved approval by the U.S. Food and Drug Admin-istration in November 2010 for prevention of skeletal-related events in patients with bone metastases from solidtumors.

Conclusions

The early findings of an essential role of RANKL inphysiologic osteoclastogenesis was the fundamentalrationale for targeting this pathway as a treatment forskeletal complications in cancer, and a therapeutic anti-body, denosumab, has been successfully developed in theclinic for this application in patients with solid tumorbone metastases. As preclinical experiments have shown,the interference with the bone microenvironment andpotent reduction in bone resorption with a RANKL inhib-itor also has potential utility in the inhibition of earlymetastatic colonization. Consistent with this hypothesis,denosumab was recently shown to be effective in delayingdevelopment of bone metastasis in men with CRPC (70),and this study represents the first large randomized studyto show that targeting the bone microenvironment hasthis effect in patients with cancer. This hypothesis is alsocurrently being addressed in women with early-stagebreast cancer at high risk of disease recurrence to deter-mine whether denosumab in combination with standard-of-care adjuvant and/or neoadjuvant cancer treatmentwill improve bone metastasis–free and disease-free sur-vival compared with standard-of-care treatment alone(71).

Although more recent preclinical basic research inrodents has indicated that RANKL can promote a spectrumof effects relevant to cancer initiation, progression, andmetastasis via direct effects on either tumor cells or themammary epithelium, significant gaps remain in the trans-lation of these more recent findings to the clinic. Theprominent role of RANKL as a paracrine effector of proges-terone action in the mouse mammary epithelium clearlyhas implications in humans, given the potential role forprogesterone specifically as a risk factor in human breastcancer, as supported by its well-established mitogeniceffects in the breast (72) and by large epidemiologic studies(73). However, fundamental differences exist in the anat-omy and hormone responsiveness of the rodent mammarygland compared with the primate, which may limit theutility of the preclinical findings. Data linking the RANKLpathway (RANKL, RANK, and OPG) expression with hor-mone exposure and breast density and proliferation inhumans, or in a suitable nonhuman primate model, areneeded to show relevance of the progesterone and RANKLpathway association. To address any association of theRANK/RANKL pathway with cancer risk or progression,genome-wide association studies or analysis of mRNA orprotein expression may be helpful, but each approach hasits limitations. Using a candidate gene approach, a recentgenetic association study has associated a single nucleotidepolymorphism in the RANK gene with breast cancer risk(74). In addition to progesterone, multiple other potentialstimuli of RANKL in cancer may exist (e.g., PTHrP, prolac-tin, tumor stromal cells, infiltrating T lymphocytes), andfurther studies are necessary not only to verify the manydiverse sources of RANKL with well-validated methodolo-gies but also to determine whether any of these RANKLsources are associated with disease outcome. Likewise, itwill be crucial to define any tumor subpopulations thatexpress RANK, again using well-validated and specificapproaches, and to relate RANK expression with addi-tional disease outcomes. Recent analysis of RANK mRNAexpression in human breast cancer biopsies indicatesrelatively increased expression in the basal tumor subtypeand an association of higher RANK mRNA expressionwith poor survival (53). Finally, the identification oftumor-specific (or mammary epithelial-specific) biomar-kers of a RANKL response would aid in the translationalevaluation of this drug in extraskeletal tissues and, ulti-mately, the testing of the current preclinical hypotheses inpatients.

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 andGeoff 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.






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