pathogenesis of osteoblastic bone metastases from prostate cancer

Pathogenesis of Osteoblastic Bone MetastasesFrom Prostate CancerToni Ibrahim, MD, PhD; Emanuela Flamini, BSc; Laura Mercatali, BSc; Emanuele Sacanna, BSc; Patrizia Serra, BSc;

and Dino Amadori, MD

Prostate cancer is the second leading cause of cancer-related death in men. A typical feature of this disease is its

ability to metastasize to bone. It is mainly osteosclerotic, and is caused by a relative excess of osteoblast activity,

leading to an abnormal bone formation. Bone metastases are the result of a complex series of steps that are not yet

fully understood and depend on dynamic crosstalk between metastatic cancer cells, cellular components of the bone

marrow microenvironment, and bone matrix (osteoblasts and osteoclasts). Prostate cancer cells from primary tissue

undergo an epithelial-mesenchymal transition to disseminate and acquire a bone-like phenotype to metastasize in

bone tissue. This review discusses the biological processes and the molecules involved in the progression of bone

metastases. Here we focus on the routes of osteoblast differentiation and activation, the crosstalk between bone cells

and tumor cells, and the molecules involved in these processes that are expressed by both osteoblasts and tumor

cells. Furthermore, this review deals with the recently elucidated role of osteoclasts in prostate cancer bone metasta-

ses. Certainly, to better understand the underlying mechanisms of bone metastasis and so improve targeted bone

therapies, further studies are warranted to shed light on the probable role of the premetastatic niche and the involve-

ment of cancer stem cells. Cancer 2010;116:1406–18. VC 2010 American Cancer Society.

KEYWORDS: osteoblasts, osteoclasts, prostate cancer, bone, metastasization.

Solid tumors, such as breast and prostate cancer, have an affinity to metastasize to bone, causing osteolysis and abnor-mal bone formation. Bone metastasis starts with the tropism of cancer cells to the bone through specific migratory andinvasive processes. Once in the bone marrow, metastatic cells are able to survive and grow. Here, they actively interactwith bone marrow stem cells and hematopoietic progenitors in the so-called metastatic niche, where they acquire a bone-like phenotype. This leads to the formation of bone lesions (lytic or osteoblastic), obtained through reciprocal paracrineamplification and cell-to-cell communication with bone cells. The complex molecular pathogenesis mechanisms of bonemetastasis offer several potential targets for prevention and therapy.

Prostate cancer is the second leading cause of cancer-related death in men, and a typical feature of this disease is itsability to metastasize to bone. Indeed, it has been estimated that >80% of men who die from prostate cancer developbone metastases.1,2 Although most bone metastases from prostate cancer have been classified as osteoblastic, based on theradiographic appearance of lesions, it is clear that bone resorption and bone formation are dysregulated.3-5

The Metastatic Process: From Primary Tumor to Growth in Bone Tissue

The first step in metastasization is the acquisition of motility and invasiveness; capabilities that are not compatible with nor-mal tissue. Cancer cells must therefore shed many of their epithelial characteristics, detach from epithelial sheets, andundergo a drastic alteration, a process referred to as the epithelial-mesenchymal transition.6 Acquisition of this invasive phe-notype is reminiscent of that which occurs during early embryonic development.7 In malignancy, genetic alterations and thetumor environment can both induce epithelial-mesenchymal transition (EMT) in tumor cells. The important steps that facil-itate metastasis appear to be reversible,7 and cannot be explained solely by irreversible genetic alterations, indicating the exis-tence of a dynamic component to human tumor progression. In cancer, although the PI3K/Akt pathway is the primaryinducer of epithelial-mesenchymal transition, the Wnt/B-catenin, Notch, Ras, integrin-linked kinase, and integrin signaling

DOI: 10.1002/cncr.24896, Received: May 14, 2009; Revised: July 2, 2009; Accepted: July 17, 2009, Published online January 27, 2010 in Wiley InterScience

(www.interscience.wiley.com)

Corresponding author: Dino Amadori, MD, Osteo-oncology Center, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori, via Piero Maroncelli 40,

47014 Meldola (FC), Italy; Fax: (011) 39 0543 739249; [email protected]

Osteo-oncology Center, Scientific Institute of Romagnolo for the Study and Cure of Tumors, Meldola, Italy

We thank Dr. Ian Seymour for help with editing the article.

1406 Cancer March 15, 2010

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pathways are also involved.8-11 The main characteristics ofepithelial-mesenchymal transition in prostate cancer cells,and the complex molecular interactions that facilitate thisprocess, are summarized in Figure 1.

After epithelial-mesenchymal transition, prostatecancer cells must go through a multistep process to meta-stasize to bone, which involves dislodgement from a pri-mary site, survival in the circulation, binding to theresident cells in bone, and survival and proliferation in thebone and bone marrow.12-18 The dissemination of pros-tate cancer cells may take place early in disease progressionwith tumor cells preferentially engaged in the bone mar-row, and a subset of cells surviving and evolving into clini-cally apparent disease. These cells then enter a period ofdormancy in which they either stop proliferating, or pro-liferate at a reduced rate before showing evidence of me-tastasis; a process that can sometimes exceed 10 years.19-23

However, in some situations, there is at least 1 further andcrucial event that takes place, the trigger that reactivatestumor cell dormancy. However, the mechanisms thatfacilitate this process remain unknown.

Circulating prostate cancer cells preferentiallyadhere to bone marrow endothelial cells and then migrate

through the endothelial layer.24 This process involves avariety of adhesion molecules (eg, selectins, integrins, andcadherins) present on the surfaces of endothelial and met-astatic prostate cancer cells. Cell adhesion and migrationare mediated in part by many integrin-extracellular matrixinteractions, especially those involving integrin avb3,whose expression is enhanced by stromal cell-derived fac-tor-1 (SDF-1).25,26 Several lines of evidence suggest thatSDF-1 contributes to the pathogenesis of prostate cancermetastases. Inhibition of chemokines reduces the in vitroproliferation of PC-3 cells. Expression of the SDF-1 re-ceptor, CXCR4, has been detected in prostate cancer cells,and SDF-1 has also been reported to increase their migra-tion capacity.1,27 This evidence implies that several boneparacrine factors regulate the expression of adhesion mole-cules in disseminated prostate cancer cells, and supportsthe interaction between cancer cells and resident bonecells, thereby contributing to the tropism of circulatingprostate cancer cells toward bone. Cadherin 11 has beenimplicated in this process because of the finding that it isoverexpressed in prostate cancer bone metastases but notin normal tissue or nonmetastatic cancer. Furthermore, ina mouse model, injection of PC3 cells did not lead to any

Figure 1. Genetic modifications of prostate cancer cells from primary tumors to bone tissue are shown. UPA indicates urokinase-type plasminogen activator; PTHrP, parathyroid hormone-related protein; IGFBF, insulin-like growth factor-binding protein; TGF-b,transforming growth factor-b; IGF, insulinlike growth factor; BMP, bone morphogenetic protein; ET-1, endothelin 1; PGDF, platelet-derived growth factor; FGF, fibroblast growth factor; VEGF, vascular endothelial growth factor; RANKL, receptor activator of thenuclear factor-jB ligand.

Bone Metastases and Prostate Cancer/Ibrahim et al

Cancer March 15, 2010 1407

bone metastasis if cadherin 11 was silenced.28 Monocytechemotactic protein-1 (MCP-1) is chemotactic for pros-tate cancer cells, and the expression of its receptor, CCR2,correlates with pathologic stage.29-31 Lu et al reported thatthe activation of MCP-1/CCR2 axis promotes prostatecancer growth in bone.32 Once metastatic prostate cancercells arrive in the bone, they are stimulated by growth fac-tors present in the noncellular fraction of bone marrow,improving their interactions with the resident bone cells.

Metastasis suppressor genes encode a class of pro-teins that block the metastatic process without affectingprimary tumor progression. These genes are rarelymutated, but are instead thought to be controlled by epi-genetic events such as gene methylation. The first metasta-sis suppressor gene characterized, Nm23,33 whenoverexpressed in metastatic cell lines, has been shown todecrease metastatic competency in vivo, and motility andinvasion ability in vitro.33-36 There is mounting evidencethat many metastasis suppressor genes, including KAI1,CD44, and MAPK4, are able to block metastasis in pros-tate cancer cell lines, and several clinical studies have alsosupported their involvement.37-39 KAI1, the first prostatecancer metastasis suppressor gene identified, is down-regulated in both metastatic and high-grade prostate can-cer.37,40 Although the mechanism of suppression is stillunclear, it is believed that KAI1 interacts with severalmembrane proteins implicated in metastasis progression,such as E-cadherin, beta1 integrins, and epidermal growthfactor receptor. CD44 is a widely expressed cell adhesionprotein, and its down-regulation has been correlated toprostate cancer grade and metastatic stage.38,41,42 Similarresults have also been reported forMAPK4.39

Osteoblast Function and Their Regulation

The same regulatory pathways essential for bone develop-ment and remodeling are probably also involved in pros-tate cancer bone metastases. Osteoblast growth anddifferentiation are regulated by complex signaling path-ways mediated by growth factors such as bone morphoge-netic proteins (BMPs), insulinlike growth factor (IGF)-Iand IGF-II, transforming growth factor-b1 (TGFb1) andTGFb2, fibroblast growth factor (FGF), platelet-derivedgrowth factor (PDGF), and Wnt.43-48 Moreover, otherfactors, such as vascular endothelial growth factor(VEGF), are thought to have an indirect action on osteo-blast function through their modification of the bonemicroenvironment.

The transcription factor RUNX2 (also called corebinding factor 1) stimulates BMP2 and FGF, and is essen-

tial for osteoblast differentiation.49 In mice, inactivationof RUNX2 results in a skeleton made entirely of cartilage,indicating that RUNX2 is necessary for bone formation.49

Another transcription factor, Osterix, is believed to con-trol osteogenesis, based on the evidence thatOsx null micedo not undergo any bone development.50 Furthermore,b-catenin, a factor that mediates the Wnt signaling path-way, has also been implicated in osteogenesis regulation.A Wnt coreceptor, which regulates bone formationthrough b-catenin, interacts with Wnt by forming a com-plex with the Wnt receptor, Frizzled.51 This complex acti-vates the canonical Wnt signaling pathway, in turnstabilizing free cytoplasmic b-catenin, which is then trans-located to the nucleus. Here, it heterodimerizes with tran-scription factors of the lymphoid enhancer-bindingfactor/T-cell factor (LEF/TCF) family to regulateunknown genes that control bone formation.52,53

Interactions between prostate cancerand bone cells

Histopathologic analysis of prostate cancer bonemetastases typically shows a large number of osteoblastsadjacent to prostate cancer cells. In contrast, osteoblastsare almost always absent in normal bone or in bone metas-tases from other cancers (such as breast, lung, and kidney),which largely contain osteoclasts.51 The increase in pros-tate cancer bone-forming activity gives rise to a wovenbone, characterized by an osteosclerotic appearance dis-tinct from the typical lamellar structure seen in normalbone. These lesions are associated with an increase inbone mass at the lesion site, and often have an elevatedosteoid surface area, osteoid volume, and mineral apposi-tion rate.54-56

Supporting the observation that prostate cancerbone metastasis is associated with increased osteoblast ac-tivity, serum levels of osteoblast proliferation markers,such as bone-specific alkaline phosphatase and type 1 pro-tocollagen C-propeptide, have been observed to be higherin metastatic prostate cancer patients.57 The increasednumber of osteoblasts often present in the woven bonesurrounding the metastatic lesions suggests a correspond-ing increase in osteoblast proliferation and differentiation.This may contribute to prostate cancer cell survival andinvasiveness by providing abundant extracellular matri-ces.58 In vivo experiments by Gleave et al59 confirmedosteoblast involvement in prostate cancer bone metastasis,showing that factors secreted by bone fibroblasts, but notother cell types, accelerate prostate cancer growth. Invitro, Fizazi et al60 established the stimulation of prostate

Review Article


cancer cells by the culture medium of osteoblast-like celllines. In these studies, primary mouse osteoblasts, cocul-tured with MDA prostate cancer 2b cells, led to cancercell proliferation, hinting at the possibility of paracrineinteractions between osteoblasts and the prostate cancercell line.60 In addition, osteoblast-conditioned mediumwas found to stimulate prostate cancer cells into produc-ing matrix metalloproteinase-9 (MMP-9) and urokinase-type plasminogen activator (uPA), while at the same timeincreasing the rate of prostate cancer proliferation.61 Fur-thermore, newly formed bone may secrete chemoattrac-tants that favor migration and enhance the invasioncapability of prostate cancer cells. Osteopontin, osteonec-tin, and bone sialoprotein can also modulate the proper-ties of prostate cancer cells.62 Osteonectin increases themigration and invasiveness of PC3 and DU145 cells,63

and bone sialoprotein facilitates the attachment of cancercells to the bone and enhances their metastatic potential.64

These observations indicate that bone-derived factors areable to initiate a series of cellular events that collectivelyhelp in cancer cell proliferation. Indirect evidence has alsoshown that this effect is specific to prostate cancer cells,because conditioned medium from osteoblasts does notenhance the growth of other cancer cell types.65 Clinicaltrials with specific bone-homing radiopharmaceuticalssuch as strontium-89, samarium, and rhenium, with dep-osition at sites of increased osteoblast activity and bone-matrix synthesis, have provided significant palliative bene-fits.66,67 Taken together, these observations indicate thatosteoblast inhibition has a direct effect on the pathogene-sis of osteoblastic lesions.

Receptor Activator of the Nuclear Factor-jB/RANK Ligand/Osteoprotegerin Axis

Osteoblasts also control osteoclast activity through theexpression of cytokines such as the receptor activator ofthe nuclear factor-jB (RANK) ligand (RANKL), a key ac-tivator of osteoclast differentiation, and osteoprotegerin, asoluble decoy receptor that inhibits RANKL.68,69 Anincrease in the level of serum osteoprotegerin (OPG) hasalso been established in advanced prostate cancerpatients.57,70 These findings show that osteoblasts, bymeans of their ability to control prostate cancer cell andosteoclast proliferation, operate as the ‘‘master switch’’ forprostate cancer progression in bone. Previous breast andprostate cancer studies in rodent models have proved thatRANKL inhibition decreases bone lesion developmentand tumor growth in bone.71-73 Furthermore, RANK is

expressed in prostate cancer cells and promotes invasionin a RANKL-dependent manner.74

Acquisition of a Bone-Like Phenotype

The acquisition of a bone cell-like phenotype allows pros-tate cancer cells to home, survive, and proliferate in bone.It has yet to be confirmed whether cancer cells alreadypossess osteomimetic phenotypes when they detach fromprimitive sites, or whether some of these characteristics areinstead acquired in the bone marrow. Prostate cancer cellsexpress several factors involved in normal bone develop-ment and remodeling (Fig. 1 and Table 1). These factorshave been implicated in osteoblastic lesions, either byaffecting osteoblast function directly, or by influencingbone formation indirectly through modification of thebone matrix or microenvironment.

BMPs

BMPs belong to the TGFb superfamily, and are the mostpowerful inductive bone factors enriching the bone ma-trix.75 Primary and metastatic prostate cancer have differ-ent phenotypic patterns of BMP expression, and adoptdiscrete downstream signaling pathways.76 BMPs demon-strate a clear and close relationship with the developmentand progression of primary prostate cancer, and also con-tribute to the onset and development of bone metastases.As a result, it has been postulated that BMPs play an im-portant role in the etiology of the osteoblastic bone metas-tasis phenotype. Indeed, BMP-secreting prostate cancercell cultures have been shown to promote in vitro

Table 1. Molecules Produced by Prostate Cancer CellsInvolved in Bone Metastases

Effects on Osteoblasts

Molecules Differentiation Proliferation Migration

ET1 � �

BMPs �

IGF � �

FGF � �

MDA-BF-1 � �

PDGF �

PSA � �

uPA � �

VEGF �

TGF-b �

Wnt �

PTHrP �

ET1 indicates endothelin 1; BMP, bone morphogenetic protein; IGF, insulin-

like growth factor; FGF, fibroblast growth factor; PDGF, platelet-derived

growth factor; PSA, prostate-specific antigen; uPA, urokinase-type plasmin-

ogen activator; VEGF, vascular endothelial growth factor; TGF-b, transform-

ing growth factor-b; PTHrP, parathyroid-hormone-related protein.



mineralization (potency from high to low: BMP6 >

BMP7 > BMP4).77 Furthermore, several BMPs areknown angiogenic factors that indirectly facilitate the de-velopment of bone metastases via the angiogenic route.On 1 hand, BMPs produced by prostate cancer cells areable to activate osteoblasts, leading to lesions that are pre-dominantly osteoblastic in nature. On the other hand,BMPs synthesized by osteoblasts, or released from thebone matrix, subsequently enhance the growth andaggressiveness of prostate cancer cells, which in turnincreases the production of BMPs by tumor cells.

Growth Factors

The IGF system, comprising 3 receptors, 3 ligands, and 6IGF-binding proteins (IGFBPs), is involved in the mito-genic, transformational, and antiapoptotic activities ofseveral cell types. In the prostate, this system plays an im-portant role in both normal and tumoral cells. IGF-I issecreted by the prostatic epithelium, probably in responseto growth hormones, and even in hypertrophy, high levelsof IGF-I have been reported.78 Prostate cancer cell lineshave been shown to express a range of different IGF com-ponents.79 IGF-I and II are abundant in the bone envi-ronment, and are known to stimulate osteoblasts,increasing bone matrix apposition, while at the same timedecreasing collagen degradation.80 Several studies haveshown a possible relationship between IGFs and bone me-tastasis from prostate cancer.81,82 IGF-I and II increasethe proliferative and chemotaxis activities of prostate can-cer cells in vitro.81 Moreover, the IGF-I pathway is up-regulated in prostate cancer cells metastasized to bone.82

Several in vivo studies have added further weight to thisimportant link. Serum IGF-I levels are correlated with therisk of developing prostate cancer,83 plasma IGFBP-III islower in patients with bone metastases, and IGFBP-II iselevated in prostate cancer patients.84 In addition, highIGF-I and low IGFBP-III levels are associated with therisk of developing advanced prostate cancer.85 Further-more, polymorphisms of IGF-I and cytochrome P450enzyme 19 have been reported to accurately predict bonemetastasis in prostate cancer patients.86 However, Rubinet al87 recently showed that IGF-I is neither necessary norsufficient for the osteoblastic response to prostate cancermetastases. For this reason, it is still not clear how the IGFsystem participates in the formation of osteoblastic pros-tate cancer bone metastasis, and additional studies arerequired to delineate the role of the IGF system in thisprocess.

Parathyroid Hormone-Related Proteinand Endothelin 1

The osteolytic factor parathyroid hormone (PTH)-relatedprotein (PTHrP) is a homolog of PTH that has a directaction on PTH receptors, increasing bone resorption andrenal tubular calcium reabsorption.88 In bone metastases,the release of PTHrP by cancer cells, together with otherfactors, contributes significantly to metastatic spread.89,90

PTHrP is abundant in prostate cancer bone metastases,and in these tumors osteoblastic lesions tend to predomi-nate.91 One possible explanation for this paradox is thatprostate cancer-derived PTHrP mediates the interactionsbetween the bone marrow microenvironment and pros-tate cancer, which further facilitates the establishment ofskeletal metastases and osteoblastic alterations. Liao et al92

have provided evidence supporting this opinion, report-ing that PTHrP increases osteoblastogenesis-stimulatingosteoblast progenitor cell proliferation and induces earlyosteoblast differentiation.

Another theory is that NH2-terminal fragments ofPTHrP share a strong sequence homology with endothe-lin (ET) 1, thus stimulating new bone formation throughactivation of the ETA receptor.93 As an osteoblast mito-genic factor, ET1 promotes osteoblast growth at meta-static sites.94 Nelson et al95 showed that plasma ET1concentrations are significantly higher in metastatic pros-tate cancer patients, suggesting that ET1 may be secretedby prostate cancer cells. A recent study has also suggestedthat ET1 increases osteoblast proliferation and new boneformation through activation of the Wnt signaling path-way and suppression of the Wnt pathway inhibitor,DKK1.96

uPA

Prostate cancer cells produce other factors, including uPAand prostate-specific antigen (PSA) that, although notdirectly involved in normal skeletal development, affectosteoblast function by modifying the bone microenviron-ment.97 The serine protease uPA is involved in degrada-tion of the extracellular matrix, which facilitates tumorcell invasion by increasing osteoblastic prostate cancerbone metastases. The pathogenetic model of osteoblasticprostate cancer metastases can be summarized as follows.The neoplastic cell produces high molecular weight uPA(HMW-uPA) that binds uPA receptor (uPAR) on the tu-mor cell surface and exerts its proteolytic action, leadingto tumoral invasion. HMW-uPA is then split into lowmolecular weight uPA and amino terminal fragment,which binds uPAR on the osteoblast cell surface, helping

Review Article


to trigger the cascade of events leading to osteoblast prolif-eration and activation. Furthermore, uPA can cleave andactive TGFb, which is produced in a latent form by osteo-blasts. TGFb regulates osteoblast and osteoclast differen-tiation as well as the growth of tumor cells themselves. Inaddition, uPA stimulates osteoblast proliferation, prob-ably by hydrolyzing IGFBPs, thereby increasing free IGFlevels.98-100 PSA belongs to the kallikrein serine proteasefamily, is secreted by prostate cancer cells, and is usedroutinely as a marker of prostate cancer progression. PSAnot only cleaves PTHrP to release osteoblastic PTHrPfragments, but also activates osteoblast growth factorssuch as TGFb.100 Consequently, PTHrP degradation byPSA decreases bone resorption,101 allowing the osteo-blast response to predominate. As with uPA, PSA canalso cleave IGFBP3, thereby freeing up IGF-I to bind toits receptor and stimulate osteoblast proliferation.102

Other Molecules

The growth factor MDA-BF-1 has recently been identi-fied in the supernatant of bone marrow specimens frompatients with prostate cancer bone metastases.103 Thissecreted form of the ErbB3 growth factor receptor isexpressed in prostate cancer cells that metastasize to bone,but not in the primary tumors of patients with localizeddisease, nor in prostate cancer cells that metastasize toother sites. Its function is mediated by an osteoblast-expressed receptor,104 and through specific interactionsbetween prostate cancer cells and bone. Scientific litera-ture is rich in papers describing new proteins that seemto be involved in prostate cancer bone metastases. One ofthese is the tyrosine kinase receptor c-Kit, and its ligandstem cell factor (SCF).105 In an experimental model,prostate cancer bone metastasis was found to stronglyexpress c-Kit, but prostate cancer cell lines were c-Kitnegative. In addition, immunohistochemical analysisshowed a higher expression of this protein in bone metas-tasis compared with primary tumors, and SCF was oftenoverexpressed.105

Prostate cancer cells produce a range of additionalosteoblast-regulatory growth factors, including PDGF,FGF, and VEGF. The dimeric polypeptide growth factorPDGF has 2 subunits, A and B, that form AA, BB, andAB isoforms. The BB isoform is known to be a potentosteotropic factor, contributing to the formation of osteo-blastic lesions through the promotion of osteoblast migra-tion and proliferation.106,107 Although FGF1 (acidic) andFGF2 (basic) are both able to increase osteoblast prolifera-tion, only FGF2 suppresses osteoclast formation.108 It is

clear that further investigations are necessary to fullyunderstand the complex FGF interplay in bone metasta-sis. VEGF appears to have both a direct and an indirecteffect on bone growth by activating osteoblasts and pro-moting angiogenesis, respectively.77,109 The Wnt familyligand has been reported to be up-regulated in the tumorcells of advanced metastatic prostate cancer patients.110

Furthermore, Wnt production can act in a paracrine wayto induce osteoblastic bone metastases. As mentioned pre-viously, the Wnt signaling pathway can be blocked by theWnt antagonist, DKK1. This molecule is usuallyexpressed early in the development of skeletal metastases,resulting in the masking of osteogenic Wnts, which favorsmetastatic site osteolysis. As metastasis progresses, adecrease in DKK1 expression unmasks Wnt-mediatedosteoblastic activity, leading to osteosclerosis.111,112

The Role of Osteoclasts

Although prostate cancer bone metastasis is osteoblastic innature, as revealed by radiographic imaging, the presenceof osteolytic-osteogenic bone lesions in osteoblastic casesmay account for the increase in observed fractures.113 Keysteps in the successful establishment of prostate cancerbone metastases appear to be osteoclast formation andbone resorption, followed by the release of several growthfactors from the bone matrix.114 However, the mecha-nisms by which prostate cancer cells promote this processremain unclear. It has been suggested that RANKL, inter-leukin (IL) 6, IL-8, and C-C chemokine ligand 2 mediateosteoclast formation from human mononuclear bonemarrow cells.31,32,99 Furthermore, prostate cancer cellshave been shown to secrete factors that promote humanbone marrow mononuclear cell osteoclastogenesis.31

Other authors have observed that IL-6 affects fusion butnot resorption, and that osteoclastogenesis in prostatecancer bone metastasis is induced by a RANKL-independ-ent mechanism. A possible hypothesis put forward is thatprostate cancer cells, by producing PTHrP, can facilitateboth osteoclastogenesis and osteoblastogenesis.115

Another supposition is focused on C-C chemokine ligand2, which may serve 2 distinct roles in the developmentand promotion of prostate cancer: a direct effect on epi-thelial prostate cancer cells, and an indirect effect onosteoclasts and endothelial cells at the metastatic site,thereby supporting tumor growth. The presence of C-Cchemokine ligand 2 in the bone microenvironment maycontribute to prostate cancer growth by initiating osteo-clastogenesis and angiogenesis, creating a favorable nichefor metastases.116



The Vicious Cycle of OsteoblasticBone Metastasis

The complex interactions between tumor cells, bone cells,and the bone matrix constitute a vicious cycle of osteo-blast-mediated bone metastasis (Fig. 2). In the earlystages, prostate cancer cells produce osteogenic factorssuch as PDGF, ET1 and BMPs, which activate osteo-blasts. Once differentiated from their progenitor cells,osteoblasts deposit a new matrix for bone formation.However, this unmineralized matrix provides a more fer-tile soil for tumor cells, enriched with growth factors andnoncollagen proteins. Moreover, newly formed bone mayprovide additional factors to attract prostate cancer cells,allowing them to survive and proliferate in the bone envi-ronment, which in turn further activates osteoblasts.However, the osteoblastic nature of bone metastases mayrepresent a double-edged sword regarding the develop-ment of lesions; the initial increase in bone volume may

limit the space available to cancer cells, and therefore helpto confine the tumor. This may explain why osteoblasticprostate cancer metastases seem to progress more slowlythan osteolytic metastases from other tumors. In addition,especially at the beginning of the process, tumor-derivedfactors and RANKL-secreting osteoblasts can both acti-vate osteoclasts, leading to some level of bone resorption,which subsequently creates more space for dominantosteoblastic lesions. Cytokines released from the bone ma-trix during bone resorption can enhance this vicious cycleby facilitating the continued proliferation of prostate can-cer cells and osteoblasts. Regrettably, as soon as the meta-static disease enters the osteoclastic stage, diseaseprogression is rapid, and survival times are short.54,117,118

Clinical Applications

A deeper understanding of the molecular mechanisms ofprostate cancer bone metastases will hopefully identify

Figure 2. The vicious cycle of osteoblastic bone metastasis is shown. (A) Prostate cancer cells secrete osteogenic growth factors,activating osteoblasts to deposit new bone matrix. (B) Osteoblasts secrete a range of additional factors such as insulin-likegrowth factor (IGF), fibroblast growth factor (FGF), and transforming growth factor-b (TGFb). (C) These factors attract prostatecancer cells, further enhancing their proliferation and growth. ET1 indicates endothelin 1; PDGF, platelet-derived growth factor;BMP, bone morphogenetic protein; RANK, receptor activator of the nuclear factor-jB; RANKL, RANK ligand; OPG,osteoprotegerin.

Review Article


new biological targets for innovative drugs, besides zole-dronic acid, which has become the standard treatment forthis disease, to be used in combination with conventionaltherapies, as well as new predictive or prognostic markers.One of the most extensively studied pathways in recentyears, the RANK/RANKL/OPG axis, has been advocatedas a potential therapeutic target because it governs bonehomeostasis, both under normal physiologic conditions,and during bone metastasis progression. In preclinical andclinical trials, inhibition of RANKL with OPG reducesbone turnover markers,119 suggesting that RANKL, inaddition to having an effect on bone lysis, also has a directaction on tumor cells.120,121 The anti-RANKL monoclo-nal antibody, denosumab, has recently been tested againstzoledronic acid in a randomized prostate cancer bone me-tastases phase 2 study,122 and was found to decrease uri-nary bone turnover markers in zoledronic acid resistantpatients. Phase 3 bone metastases studies are ongoing withskeletal-related events (SREs) as the primary objective,and results should be available shortly.

Atrasentan, an ETA receptor antagonist, is beingtested in clinical trials for the treatment of prostate cancer,and it has been shown to prevent osteoblastic bone metas-tases in mouse models, as well as reduce disease progres-sion in advanced phase 2 prostate cancer trials.123,124

However, a recent phase 3 study of atrasentan versus pla-cebo reported no significant differences in disease progres-sion,125 and docetaxel/atrasentan prostate cancer studiesare still ongoing. The monoclonal antibody vitaxin bindsintegrin avb3, and androgen-independent prostate cancerclinical studies are currently in progress.126 This moleculehas demonstrated both in vivo and in vitro antitumoractivities, affecting bone resorption by impairing osteo-clast attachment, whereas osteoclast formation and multi-nucleation processes remain unaffected.

Other targets of bone metastases include Akt, cyclo-oxygenase-2, and MMP-9. Diaz et al127 recently investi-gated the Akt inhibitor Palomid 529 in vitro, and foundthat it had a synergistic effect with radiotherapy, enhanc-ing the antiproliferative effect of radiation in prostate can-cer cells, while decreasing p-Akt, VEGF, MMP-9, MMP-2, and Id-1 levels. Another drug target of MMP-9, 3,30-diindolylmethane,128,129 is an inhibitor of the DNAbinding activity of nuclear factor-jB, which mediatesthe expression of many genes such as VEGF, IL8, anduPA. Antibodies against IGF-I receptor, and IGF-I re-ceptor-specific tyrosine kinase inhibitors, have beendeveloped as anticancer agents, and some have alreadybeen tested in clinical trials against solid tumors, show-

ing promising results.130,131 These are just a few of thevast array of new drugs currently under preclinical andclinical experimentation.

Regarding the monitoring of metastatic prostatecancer, the most commonly cited markers seem to be theproducts of collagen degradation.132-135 This categoryincludes N-telopeptide (NTx) or C-telopeptide type Icollagen (CTX), amino-terminal procollagen propep-tides (PINP), and cross-linked C-terminal telopeptides(ICTP). Other common markers of osteoblastic bone me-tastases include total and bone-specific alkaline phospha-tase, serum tartrate-resistant acid phosphatase, andPSA.132,133 In a recent study of 100 patients, Klepziget al134 observed that procollagen propeptides couldreliably predict prostate cancer bone metastases. Anotherimportant study by Lein et al133 has focused further atten-tion toward procollagen propeptides, NTx, and PSA,establishing bone markers as useful tools for the predic-tion and diagnosis of SREs in patients with prostate can-cer bone metastases undergoing zoledronic acid therapy.NTx has recently undergone validation in 3 large retro-spective phase 3 trials.135 This marker was found todecline to normal physiologic concentrations within 3months, and, compared with high NTx levels, was associ-ated with a reduced risk of skeletal complications anddeath. In contrast, PSA has been reported to be a poormarker, both for identifying patient populations at highrisk of metastatic disease and for monitoring skeletal pro-gression during treatment.132 A further study by Jung etal57 tested 10 serum markers in 117 prostate cancerpatients, citing OPG and bone sialoprotein as excellentbone metastases markers in addition to being independentprognostic factors for prostate cancer-related death.

Conclusions

Cancer cells can only metastasize in organs where themicroenvironment accommodates their growth. In otherwords, the complex interactions between cancer cells andthe bone/bone marrow microenvironment are fundamen-tal to the establishment of bone metastases. Prostate can-cer cells from primary tissue undergo epithelial-mesenchymal transition to disseminate and acquire abone-like phenotype to metastasize in bone tissue. Afterthe arrival of prostate cancer cells in bone, their crosstalkwith the bone microenvironment facilitates bone metasta-ses, and defines their osteoblastic pattern. Finally, recentdiscoveries on the mechanisms of bone metastasization,for example epithelial-mesenchymal transition and osteo-mimicry, will certainly help to improve bone target



therapies. Without doubt, there is an urgent need for fur-ther work, particularly on the potential role of the preme-tastatic bone niche, and on the involvement of cancerstem cells in bone metastases.

CONFLICT OF INTEREST DISCLOSURESThe authors made no disclosures.

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pathogenesis of osteoblastic bone metastases from prostate cancer

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