Molecular and Biological Mechanisms of Bone Metastasis

EAU Update Series

EAU Update Series 3 (2005) 214–226

Molecular and Biological Mechanisms of Bone Metastasis Marco G. Cecchinia,*, Antoinette Wetterwalda, Gabri van der Pluijmb, George N. Thalmanna a

Urology Research Laboratory, Department of Urology and Department of Clinical Research, University of Bern, Murtenstrasse 35, CH-3010, Bern, Switzerland b Department of Endocrinology and Urology, C4-R86, Leiden University Medical Center, Albinusdreef 2, 2333 ZA. Leiden, The Netherlands

Abstract Metastatic disease is the cause of death in the majority of cancer patients. Bone marrow is a preferential site of metastasis in breast and prostate cancer, responsible for the majority of skeletal metastases. Micrometastases are often present in the bone marrow of cancer patients and may progress to overt metastases. The survival of these cells and the development of bone metastases depend on the growth support provided by the bone microenvironment and the ability of cancer cells to adapt to this environment, often mimicking the behaviour and gene expression of cells of the bone and bone marrow environment. Experimental evidence suggests that the growth support provided by the bone microenvironment is active during bone resorption. Increased bone turnover as it occurs with hormonal deprivation, therefore, might be a risk factor for developing bone metastases. Interference with bone turnover, however, offers a novel target for preventive and adjuvant therapies. In this review possible mechanisms and factors involved in the development and progression of bone metastases, as well as the molecular, biological and physiological processes of metastases, especially to the bone, are discussed. Furthermore the role of bisphosphonates in the prevention and treatment of bone metastases is reviewed. # 2005 Elsevier B.V. All rights reserved. Keywords: Bone metastasis; Osteolysis; Osteosclerosis; Osteoblast; Osteoclast; Prostate cancer; Mammary cancer; Bisphosphonate

1. Introduction Because of the progress made in early detection and surgical treatment of the primary tumour, mortality in cancer patients is increasingly linked to metastatic disease. Bone is the second most frequent site of metastasis. Breast and prostate cancer (CaP) are responsible for the majority of the skeletal metastases (up to 70%). The molecular mechanisms of this propensity to colonise bone are still poorly understood. Bone metastases are a major cause of morbidity, characterized by severe pain and high incidence of skeletal and haemopoietic complications (fractures, spinal cord compression and bone marrow aplasia) requiring hospitalization. Treatment options are often unsatisfactory. * Corresponding author. Tel. +41 31 632 2259; Fax: +41 31 632 0551. E-mail address: [email protected] (M.G. Cecchini).

Metastasis is a multi-step process characterized sequentially by loss of intercellular cohesion, cell migration, angiogenesis, access to the systemic blood circulation (intra-vasation), survival in circulation, arrest and subsequent extra-vasation, evasion of local immune responses, and growth at distant organs [1,2] (Fig. 1). In animal models, it has been estimated that 3– 4
106 cancer cells/g of tumour can reach the bloodstream per day [3]. However, only a restricted minority of cancer cells reaching the blood possess the biological properties to survive and grow at the distant sites and, therefore, the development of metastasis is a relatively rare event [4–7]. Comprehension of the metastatic cascade has allowed to develop novel therapeutic approaches [8] that are now being evaluated in clinical trials such as the ZEUS study of the EAU. In the following we would like to discuss some of the aspects of the metastatic cascade.

1570-9124/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.euus.2005.09.006

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Fig. 1. Steps involved in cancer cell metastasis from primary tumour site to the skeleton. Epithelial mesenchymal transition (EMT) confers an invasive phenotype (loss of cell-cell adhesion, increased motility and matrix degradation) to the cancer cells. At the same time primary cancer promotes new bloodvessels formation (angiogenesis), which facilitate accession of the cancer cells to the systemic circulation (intravasation). Aggregates of cancer cells with platelets and leukocytes form cell emboli that may protect from immune reaction and facilitate arrest in the bone capillaries by a mechanical mechanism and by adhesion to bone marrow endothelium-specific cell adhesion molecules. Chemokines and bone matrix molecules mediate their entry in the bone marrow/bone microenvironment. Here the cancer cells, a, are exposed to the survival and growth support normally exerted on haematopoietic cells, b, by marrow stromal cells, namely stromal fibroblasts and tissue macrophages, c, and endothelial cells, d. In addition, bone cytokines secreted by osteoblasts, e, and osteoclasts, f, or matrix-integrated, g, released and activated during bone resorption, contribute critically to survival and proliferation of the metastatic cancer cells.

2. Mechanisms of metastases to bone marrow/bone As pointed out by von Recklinghausen more than a century ago, the first colonization and growth of cancer cells occurs in the bone ‘‘marrow’’ (BM). Accordingly, the biological and molecular characteristics of both bone and BM tissues should be considered when interpreting the pathophysiology of bone metastasis. Various mechanisms have been suggested to explain the great frequency of BM colonization by epithelial cancers [9,10]. Bone metastases are almost always multiple and the axial skeleton is more commonly involved than the appendicular skeleton. As first proposed by Batson [11] and later demonstrated in human cadavers by Franks [12], there exists a para-vertebral network of thin-walled veins that may play a role in the development of bone metastasis, e.g. by localisation and decreasing blood flow. Experimental evidence indicates that injection of tumor cells into the tail vein of nude mice may lead to the development of bone metastases [13], although the yield is low. By coinoculation of tumor cells with inductive fibroblasts and modulation of the hormonal milieu development of

spontaneous metastases can be induced, suggesting that other factors are necessary for the metastatic process [14]. It has been suggested that this affinity and distribution might be in relation to the hematopoietically active red bone marrow [15–17]. This is substantiated by the fact that when there is extensive infiltration of the red bone marrow by the tumor or bone marrow has been irradiated secondary sites of hematopoiesis may arise and become sites of metastatic growth. Finally, tumor cells can only survive when the microenvironment is favourable for their survival. In fact, a reciprocal interaction between the BM/bone microenvironment and cancer cells is fundamental for establishing bone metastatic growth. Once the tumor cell has detached from the primary tumor and entered the lymphatic or blood circulation, following processes are crucial: Vascular adhesion and extravasation. In the blood stream cancer cells may interact with platelets and leucocytes, to create aggregates or emboli. These may facilitate mechanical trapping in organ capillaries, resistance to shear stress and protection from immune cell-mediated tumour cell clearance [18].

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Cells circulating in blood or lymphatic vessels need to interact with the endothelium in order to extravasate and ‘‘home’’ in a specific tissue [19]. Endothelial cells in the various organs and tissues form a remarkably heterogeneous cell population not only in terms of morphological phenotype, but also with regard to their specialized functions and surface markers [20,21]. Cancer cells have been reported to adhere preferentially to the BM-derived endothelial cells in a variety of experimental settings [22,23]. Chemoattractive and adhesion molecules play a fundamental role in this selective retention of cancer cells in the BM vasculature. Each tissue requires a constitutively and specific combination of these mediators on the tissue specific endothelium (molecular ‘‘area codes’’ or ‘‘addresses’’) still susceptible to modification by specific stimuli, such as inflammation. During embryogenesis a set of BM-specific vascular cell adhesion molecules (VCAM), such as VCAM-1 and E-selectin [21,24–26] mediate BM colonization by haematopoietic stem cells (HSC) via the blood stream [27–29]. Cancer cells have been reported to utilize equivalent molecules to adhere to the BM endothelium [30–32]. Chemoattractive cytokines (Chemokines, CK) also attract HSC to the BM. CXCL12 (also known as ‘‘stromal derived growth factor-1, SDF-1), a CK normally secreted by BM stromal and endothelial cells [33] and by osteoblasts [34], attracts HSC expressing the corresponding receptor CXCR4 [35,36]. The functional role CK receptors CXCR4 and CCR7, expressed by a variety of osteotropic epithelial cancer cell types, in mediating their adhesion to BM endothelium [37,38] strongly supports the concept that CK are critical also in determining bone metastasis [39,40]. The major component of the skeletal tissue is mineralized matrix, rich in relatively bone-specific molecules, such as osteopontin (OPN), bone sialoprotein (BSP) and type I collagen, mediating local adhesion, motility, survival and growth actions by interaction with matrix adhesion molecules, namely integrins. Their expression is critical not only for guiding haematopoietic stem cells (HSC) to haematopoietic sites [41], but also for organ colonization by cancer cells [42,43]. Mainly the avb3 and aIIbb3 integrins seems to participate in determining the osteotropism of cancer cells [44–47]. OPN deficiency reduces experimental cancer cell metastasis to bone and soft tissues [48]. On the other hand, OPN expression by breast and CaPs correlate with bad prognosis and risk to develop bone metastasis [49–51], and forced expression of BSP or OPN down-regulation in cancer cell lines modifies their bone metastatic potential [49,52]. CD44 is a

non-integrin, ubiquitous and multifunctional surface adhesion molecule, which has been identified as a receptor for both the glycosoaminoglycan hyaluronan [53] and OPN [54]. CD44 exists as a number of different isoforms, reported to be expressed by various cancer cell types and with a well documented role in skeletal metastasis [55]. Intracellular OPN forms a complex with cell surface CD44 in migrating cells [56], induces CD44 expression in an autocrine fashion [57] and mediates hepatocyte growth factor-induced invasive cell behaviour [58]. All these functions may contribute to elucidate the functional relationship between host-derived and tumour cell-derived OPN. Taken together, the evidence above suggests that trafficking of normal HCS and metastatic cancer cells may involve similar mechanisms [59]. Micro-environmental support. Whatever the mechanisms are that determine the arrest of cancer cells at a specific target tissue, these must survive and eventually proliferate in order to progress to a clinically manifest metastasis. The ‘‘seed and soil’’ hypothesis, first advanced by Paget in 1889, postulates that the microenvironment specific for a target tissue provides a fertile ground (‘‘the soil’’), which is permissive for the survival and growth requirements of the metastatic cancer cells (the ‘‘seed’’) [17]. Since then relevant experimental evidence in experimental models of bone metastasis has supported this view [60,61]. The bone matrix producing cells, the osteoblasts, the cells embedded in the calcified matrix, the osteocytes, and the calcified matrix resorbing cells, the osteoclasts, together with the bone matrix itself constitute the bone specific microenvironment. During the dynamic phases of bone resorption and formation they contribute to development of bone metastasis locally through release and activation of survival and growth promoting factors [23,62–64] (Fig. 1). The BM stroma consists of a heterogeneous cell population, which include stromal fibroblasts, adipocytes, endothelial cells and tissue macrophages [65]. All these cellular components are a rich source of factors that are essential for the maintenance of HSC and for the expansion of the pool of haematopoietic progenitors [66]. They may also contribute to survival and expansion of cancer cells in the bone marrow (Fig. 1). Epithelial-Mesenchymal Transition (EMT) and ‘‘Osteomimicry’’. Epithelial cells are polarized. However, they can lose these epithelial features and acquire mesenchymal characteristics. This highly conserved and fundamental process is called Epithelial-Mesenchymal Transition (EMT) and enables epithelial cells, mainly during embryogenesis, to migrate to a new environment and differentiate into a distinct cell type

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by the reverse process mesenchymal-epithelial transition (MET). In cancer cells EMT confers the invasive phenotype. EMT and MET are both non cell-autonomous processes, but typically require an external stimulus to be initiated. External signals or ‘‘inducers’’ are extracellular matrix components, and soluble factors including members of the Transforming Growth Factor-b (TGF-b) superfamily, Fibroblast Growth Factor (FGF) family, Epidermal Growth Factor (EGF), Scattering Factor/Hepatocyte Growth Factor (SF/HGF), Insulinlike Growth Factor-II (IGF-II) and proteins of the Wnt and Hedgehog families.

3. Bone turnover Bone is continuously remodelled by microscopic patches of bone resorption and subsequent bone formation (Fig. 2). These two phases are in a balanced sequence and the net result is replacement of old bone with new bone, thus maintaining structural integrity of the skeleton throughout adult life. These morphological entities, together with their cellular components, are called ‘‘basic multicellular units’’ (BMU) [67]. The actual number and activity of these BMUs determine the bone turnover rate (or status) and they are under the control of mechanical stress, cytokines and hormones.

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Cells of the osteoblast lineage (osteoblasts and marrow stromal fibroblasts) play a critical role in inducing recruitment and activity of osteoclasts at the bone resorption site. Colony stimulating factor-1 (CSF-1) [68] and receptor activator of NF-kappaB ligand (RANKL) [69,70], a member of the tumour necrosis factor (TNF) family, are the two main factors released by cells of the osteoblast lineage, which are both essential and sufficient to generate osteoclasts. Osteoprotegerin (OPG), a decoy form of the RANKL receptor (RANK) normally present in the bone marrow, prevents RANKL from binding to the RANK present on the osteoclast progenitors. Therefore, it inhibits osteoclast recruitment and, consequently, bone resorption [71,72]. Most osteotropic factors, such as parathyroid hormone (PTH), 1,25(OH)2-vitamin-D3 and estrogens (E), and local cytokines, such as interleukin(IL-) 1, IL-6, IL-11 and IL-18 act indirectly on osteoclast generation by modulating RANKL expression in cells of the osteoblast lineage [62,73–75]. Systemic factors, such as PTH, estrogens, prostaglandins and local cytokines modulate osteoblast recruitment [64]. Cytokines recruit new osteoblasts at the BMU to fill the gap created by osteoclasts (Fig. 2). Most local mitogenic factors are embedded within the calcified matrix [76] and are released from the bone matrix and activated during bone resorption [77]. Therefore, the higher the bone turnover is, the higher are the sites of

Fig. 2. Cellular basis responsible for bone remodelling-turnover: the bone modelling unit (BMU). Cellular components of the BMU are the multinucleated bone resorbing cells, the osteoclasts (solid arrowheads), which first erode a ‘‘lacuna’’ in the calcified matrix, a, of a bone trabecula. In close sequence (‘‘coupling’’ phenomenon), the bone forming cells, the osteoblasts (arrows), lay down new bone matrix, yet non calcified (osteoid), b, which then becomes mineralized. At the end phase of this remodelling the surface of a new bone ‘‘package’’, c, is covered by flattened osteoblasts or ‘‘lining’’ cells (empty arrowheads) characterized by low/absent bone forming activity (‘‘resting’’ phase).

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paracrine availability of growth factors and, accordingly, the probability that the cancer cell, the ‘‘seed’’, will find a favourable ‘‘soil’’ in the bone/BM environment [8]. This view has been substantiated by experimental evidence in animal models of bone metastasis [78–80] and by clinical studies demonstrating that a high bone turnover rate in prostate and breast cancer patients increases the risk for bone metastasis [81–83]. In patients affected by CaP hormonal deprivation is used to delay disease progression. However, the increase of bone turnover that follows sex steroid deprivation may favour the development of bone metastases in the subsequent period [84,85]. Indeed, a ‘‘flare’’ phenomenon in bone scintigrams following orchiectomy has been described [86]. Prophylactic reduction of bone turnover rate may reduce the probability to develop clinically evident bone metastases from micrometastases in the bone marrow. This preventive approach might be even more justified in case of high bone turnover, either basal or induced by surgical or chemical hormonal deprivation, as recently demonstrated in an animal model of bone metastasis [87]. Serum alkaline phosphatase (sALP) and urinary hydroxyproline are common and non-expensive markers of bone formation and bone resorption, respectively, that are useful for assessing non-invasively the overall bone turnover rate in cancer patients at risk to develop bone metastasis. Recently, more sensitive and specific biochemical markers have become available. Immunoassays for bone-specific alkaline phosphatase and type I collagen propeptides are currently the most sensitive markers for assessing bone formation. Best indices of bone resorption are the immunoassay for the pyridinoline cross-links and the related peptides that can be measured in urine and, more recently, in serum [88].

evidence that osteotropic cancer cells might utilize the same pathways by mimicking HSC for local growth support [92,93].

4. Marrow haematopoiesis

6. Mechanisms of osteolytic metastasis

More than a century ago Paget already observed that the appendicular skeleton is very rarely or almost never affected by cancer metastasis suggesting that the haematopoietically active marrow might be involved. There is not only an anatomical contiguity but also a close interdependence between physiological and pathological bone resorption/formation and active marrow haematopoiesis [68,89]. Emerging experimental evidence indicates spatial and temporal coincidence of sites of bone formation and sites of active haematopoiesis and defines the osteoblast as having a major role in supporting expansion of HSC into the different haemopoietic lineages [90,91]. There is a growing body of

There is no direct evidence that tumour cells have the intrinsic ability to resorb bone and that osteoclasts are required for tumour-induced osteolysis and tumour growth from osteoclast-deficient mice [97]. Parathyroid hormone-related peptide (PTHrP) is produced by most solid osteotropic cancers [98,99] and plays a major role in the development of the osteolytic features of their bone metastatic lesions. In breast cancer PTHrP expression is significantly higher in bone metastatic tumour samples than in paired primary tumour samples [100–102]. Whether the bone microenvironment induces cancer cells to express PTHrP or whether cells that metastasize to

5. Histopathological features of bone metastases The bone-lysing (osteolytic) or the bone-forming (osteosclerotic or osteoblastic) features of bone metastasis are already apparent on plain radiographs as radio-transparent or radio-opaque bone lesions, respectively. In the case of osteosclerotic lesions the histopathological analysis shows in the area adjacent to the cancer cells an increased number of plump (active) osteoblasts lining an excess of calcified bone matrix. The increased uptake of bone scanning agents at the bone metastatic site reflects this increased osteoblast activity. In the case of osteolytic lesions, there are only flattened (inactive) osteoblast (lining cells), but numerous osteoclast actively eroding the relatively scarce bone matrix. Bone regions affected by either osteolytic or osteosclerotic lesions are more prone to pathological fractures due to architectural distortion deposition of bone of the woven (immature or embryonal) type, which is much less mechanically competent than the lamellar (mature) type. The two tumours that most commonly metastasize to bone, breast and CaP, develop different types of bone lesions. The majority of patients with breast cancer develop osteolytic lesions [94], whereas in CaP osteosclerotic lesions are predominant [95,96]. On close analysis different degrees of mixed osteolytic and osteosclerotic lesions may be found, as osteosclerotic lesions require bone resorption to initiate the osteoblastic reaction.

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bone have an intrinsic higher PTHrP expression is unclear [103,104]. Expression of PTHrP by cancer cells clinically correlates with clinical outcome and probability to develop bone metastasis. PTHrP is responsible for the humoral hypercalcemia of malignancy. PTHrP stimulates osteoclast generation by inducing RANKL in cells of the osteoblast lineage and, at the same time, by down-modulating osteoprotegerin (OPG) in the same cells [105] (Fig. 3). As a result, osteoclast bone resorption is increased with consequent release and activation of matrix-integrated growth factors, especially TGF-b, BMPs and IGFs, stimulating both tumour growth and further secretion

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of PTHrP, thus establishing a ‘‘vicious cycle’’ (Fig. 3) [106]. Interleukin-8 (IL-8) has been also shown to participate to the osteolytic features of breast cancer bone metastases [107,108]. The concept of the ‘‘vicious cycle’’ has important therapeutic consequences: Inhibition of bone resorption should interrupt this vicious cycle and thus may preserve bone mass, prevent pathological bone fractures, and even arrest tumour progression in the bone metastasis [109].

7. Mechanisms of osteosclerotic (osteoblastic) metastasis

Fig. 3. The ‘‘vicious cycle’’ hypothesis of osteolytic metastasis. Reciprocal interactions between cancer cells and bone cells, the osteoclasts and the osteoblast, determines not only osteoclast activation and subsequent bone resorption, but also growth progression of the cancer cells. Production of parathyroid hormone-related peptide (PTHrP) by cancer cells induce osteoblasts to produce receptor activator of nuclear factor-kB ligand (RANKL) and to down-regulate osteoprotegerin (OPG). This results in an increase in osteoclast recruitment from bone marrow progenitors and excess of bone resorption (osteolysis). The osteolytic process releases and activates matrixintegrated growth factors, including transforming growth factor-b (TGF-b), bone morphogenetic proteins (BMPs) and insulin-like growth factors (IGFs), stimulating further cancer cell proliferation and PTHrP secretion and, thus, establishing a vicious cycle. Other cytokines, such as interleukin(IL-) 1, IL-6, IL-11 and IL-18 (not shown) act indirectly on osteoclast generation by modulating RANKL expression in cells of the osteoblast lineage.

The mechanisms determining the exaggerated osteoblast response in osteosclerotic bone metastases are still poorly understood. It is a common concept that the excess production of mineralized bone matrix adjacent to the metastatic tumor cell deposit is due to an increased secretion of factors inducing proliferation and differentiation (recruitment) of osteoblast progenitors by the metastatic cancer cells (Fig. 4). On the other side, absent or greatly reduced expression of bone resorbing cytokines may also concur to the osteosclerotic feature of the bone metastatic lesion. Factors that directly modulate proliferation and differentiation along the osteoblast lineage activate primarily transcription factors such as core binding factor-1 (cbf-1 or RUNX2) [110–112], osterix [113], and b-catenin [114]. Paradigmatic molecules regulating directly osteoblast generation are members of the TGF-b superfamily such as TGF-b1 [115], TGF-b2 [116] and the BMPs [117]. BMPs have been postulated to play an important role in osteoblastic metastasis [63]. Endothelin-1 (ET-1), a powerful vasoconstrictor [118], is also a direct mitogen for osteoblast progenitors in vitro [119] and in vivo [120,121]. Patients with osteoblastic metastasis from CaP have high serum levels of ET-1 [122]. In an animal model of osteoblastic bone metastasis, administration of an ET-1-selctive receptor antagonist resulted in both decreased tumour burden and osteosclerotic features of the bone lesions. Interestingly no effect on the tumour burden of the primary tumour was observed [123]. This indicates that inhibition of ET-1 osteoblast stimulating activity may have negative consequences on tumour growth and suggests that a vicious cycle may also occur in osteoblastic metastasis [64,123]. IGF-I and IGF-II, FGF1 and FGF-2 and plateletderived growth factor (PDGF-BB), have also been

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implicated as direct stimulators of osteoblast recruitment in osteosclerotic bone metastases [23,62,109]. The canonical Wnt signalling pathway has recently been demonstrated to be involved in determining bone mass [124–126]. This effect is exerted by mediating the anabolic effects of mechanical and PTH stimulation on osteoblasts [127–129]. Upregulation of the Wnt family ligands Wnt-1 [130] and Wnt-11 [131] has been described in tumour specimens from patients with advanced CaP and in CaP cell lines, respectively. Interestingly, in myeloma, which is characterized by osteolytic lesions with extreme suppression of osteoblastic activity, Wnt signalling is inhibited by the extracellular Wnt-antagonist dickkopf (DKK-1) expressed by the tumour cells [132]. This further emphasizes the role of Wnt signalling in osteoblast activation. Future research will clarify the role of Wnt signalling in the development of bone metastasis. Factors that indirectly activate osteoblast generation or induce microenvironmental changes, like VEGFinduced angiogenesis [133], favour osteoblast function [134] or inactivate osteoclast generation [109]. All the factors acting on osteoblast progenitors induce terminal differentiation [113,135–137]. CaP cells also produce proteases that may indirectly influence osteoblast function. Prostate-specific antigen (PSA), a serine protease of the kallikrein family [138], can cleave PTHrP, thus allowing the osteoblastic reaction to predominate by decreasing bone resorption [139,140]. PSA also cleaves insulin-like growth factor binding protein-3 (IGFBP-3) which increasing the osteoblast stimulating activity of IGFs [141]. Urokinase-type plasminogen activator (uPA), another protease, can also cleave IGFBP-3 [142,143].

8. Therapeutic interference with the bone microenvironmental support

Fig. 4. Pathophysiology of osteosclerotic (osteoblastic) bone metastasis. Normally, the amount of bone removed by osteoclast resorption is replaced by an equal amount of newly formed bone, keeping the bone mass in perfect balance. Production of factors, such as bone morphogenetic proteins (BMPs), transforming growth factor-b (TGF-b), endothelin-1 (ET-1), insulin-like growth factors (IGFs), fibroblast growth factors (FGFs), platelet-derived growth factor (PDGF) and members of the Wnt family, stimulates osteoblast recruitment at physiological sites of bone remodelling. The increased number of newly recruited osteoblasts lay down an excess of bone matrix in the resorption lacuna, resulting in a net increase in the local bone mass adjacent to the cancer cell deposits. Absence of production of factors stimulating osteoclast generation by the cancer cells favours this imbalance.

From the considerations above it derives that pharmacologic suppression of bone turnover should interfere with its growth support and, thus, prevent development and progression of bone metastases. Bisphosphonates (BP) are non-hydrolyzable pyrophosphate analogues that exert a strong inhibitory effect on osteoclastic bone resorption and exclusively accumulate in bone [144]. In vitro, BP have been reported to exert a direct antiproliferative and pro-apoptotic effect also on cancer cells [145–147], to interfere with cancer cell adhesion to bone matrix proteins [148,149], to inhibit matrix metalloproteinases [150,151] and cancer cell migration and invasion [152,153]. An antiangiogenic effect has

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also been described [154,155]. All these effects are possibly relevant for cancer disease [156,157]. However, in most of the cases they have been obtained at concentrations that are too high to be reached in vivo in the extracellular compartment. In vivo, in animal models of bone metastasis, BPs have been reported to reduce the tumour burden in established bone metastases, but not in soft tissue and visceral metastases. This effect seems to be mediated by induction of apoptosis in the cancer cells [158,159]. Prophylactic treatment with BP, given prior to the development of evident metastasis, resulted in a marked reduction of the number of bone metastases [159–162]. Taking advantage of the ability of whole body bioluminescent imaging [163] for monitoring noninvasively the tumour burden in vivo, we investigated in an animal model developed in our laboratory the effect of various BPs on both established bone metastases or, as a preventive treatment, before development of bone metastases [8]. Our results show that BPs do not affect the growth progression of already established bone metastases although they efficiently inhibit osteolysis. In contrast BPs administered two days before i.c. injection of cancer cells in preventive intent, significantly reduce the number of developing metastases. However, they do not affect tumour burden in the few, nevertheless developing bone metastases, which may even show accelerated growth progression in the surrounding soft tissues. These results suggest that, once bone metastases have developed, they may grow independently of the microenvironmental support and the level of bone turnover. In contrast, micrometastases depend, at least partially, on the level of bone turnover for initiating growth. In addition, these results seem to exclude a direct BP effect on tumour cells in vivo. Taken together, the data above suggest that interference with the bone microenvironmental support, namely inhibition of bone turnover, is useful for inhibiting early developmental steps of bone metastasis. In clinical practice, BPs have been widely used to control skeletal complications (bone metastasis and humoral hypercalcemia of malignancy) in various neoplastic diseases. In established bone metastases, BP reduce the number of skeletal related events [164–168]. Other clinical studies have also shown that BP may prevent development of new bone metastatic foci in patients either with bone metastasis already

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present or with no bone metastasis at the beginning of the treatment [169,170]. However, a similar study failed to demonstrate a reduction in the number of metastases [171]. Three clinical trials have primarily focused their investigation on the possibility that BP may prevent the development of metastases in women affected by mammary carcinoma, but free of bone metastases at the moment of diagnosis. In two of these studies [172,173], the preventive administration of BP reduced significantly the number of patients developing bone metastases and the number of the bone metastases. In one study [173] there was also a significant reduction of the number of visceral metastases. However, in the third study [174] BP treatment did not prevent the development of bone metastases and seemed even to increase the development of non-skeletal metastases. In CaP similar prevention trials are ongoing. Atrasentan is a small molecule that blocks the receptor that mediates the effects of endothelin-1 (ET-1). ET-1 is a potent mitogen for prostate cancer cells and osteoblasts alike, and modulates nociception. In a prospective randomized, placebo-controlled Phase II trial atrasentan at two different doses (2.5 mg and 10 mg po daily) delayed time to progression (TTP) and provided adequate analgesia in patients with metastatic HRPC [175]. A Phase III trial with atrasentan has completed data accrual and results are awaited. Other immuno- and gene therapeutic studies are ongoing, as well as studies with novel compounds such as receptor tyrosine kinase inhibitors, and antibodies against PTHrP.

9. Conclusions Bone metastases are an important source of morbidity and mortality in patients with prostate cancer. The survival of prostate cancers cells and the development of bone metastases depends on the growth support provided by the bone microenvironment and the ability of cancer cells to adapt to this environment. Increased bone turnover may increase local growth support. Interference with this growth support may allow to develop new therapeutic approaches with compounds such as bisphosphonates or combinations with other strategies.

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CME questions Please visit www.uroweb.org/updateseries to answer these CME questions on-line. The CME credits will then be attributed automatically. 1. Which of the following statements concerning androgen ablation therapy and bone metastases is true: A. Androgen ablation therapy decreases bone turnover B. Androgen ablation therapy has no influence on bone turnover C. Androgen ablation increases bone turnover D. Androgen ablation therapy does not cause osteoporosis 2. Which of the following statements on bisphosphonates (BPs) is wrong: A. BPs have a strong inhibitory effect on osteoclasts B. BPs do not accumulate in bone C. BPs have an anti-proliferative and anti-apoptotic effect D. BPs are used to control skeletal complications

3. Which of the following statements concerning bone metastases is wrong: A. The appendicular skeleton is frequently involved B. Prostate cancer metastases are predominantly osteoblastic C. The venous plexus along the vertebral column is called Batson’s plexus D. Bone metastasis occur preferentially in sites of haematopoietic activity 4. Bone turnover is influenced by: (1) Estrogens; (2) Bisphosphonates; (3) Endothelin-1-Receptor antagonists; (4) Prostaglandins. A. Only 1 B. 1 and 3 C. 2 and 3 D. All answers are correct