IJC International Journal of Cancer

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1 IJC International Journal of Cancer Steps in prostate cancer progression that lead to bone metastasis Jung-Kang Jin 1,2, Farshid Dayyani 1 and Gary E. Gallick 1,2 1 Department of Genitourinary Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 2 The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX Prostate cancer is a complex disease in which metastasis to the bone is the main cause of death. Initial stages of metastasis are generally similar to those for most solid tumors; however, the mechanisms that underlie the homing of prostate tumor cells to the bone are not completely understood. Prostate cancer bone metastasis is also a microenvironment-driven disease, involving bidirectional interactions between the tumor and the bone microenvironment. In this review, we discuss the current understanding of the biologic processes and regulatory factors involved in the metastasis of prostate cancer cells, and their specific properties that promote growth in bone. Although many of these processes still need to be fully elucidated, a better understanding of the complex tumor/microenvironment interplay is slowly leading to more effective therapies for patients with prostate cancer bone metastases. Prostate cancer is the second most commonly diagnosed form of cancer and the sixth leading cause of cancer-related deaths among men worldwide. 1 In patients with localized prostate cancer, the 5-year survival approximates 100%; however, in patients in whom distant metastases have occurred, the 5-year survival drops to 31%. 2 Like most other solid malignancies, prostate cancer can metastasize to distant Key words: prostate cancer, bone, metastasis, invasion, migration, osteoblast, osteoclast Abbreviations: AR: androgen receptor; BMP: bone morphogenetic protein; BMS: bone marrow stromal; CAM: cell adhesion molecule; DKK1: Dickkopf-1; ECM: extracellular matrix; EMT: epithelial to mesenchymal transition; ET-1: endothelin-1; ET A : endothelin-1 receptor subtype A; FAK: focal adhesion kinase; HBME: human bone marrow endothelium; IGF: insulin-like growth factor; IGFBP: IGF-binding protein; IGF-IR: IGF I receptor; IL: interleukin; MMP: matrix metalloproteinase; OPG: osteoprotegerin; PI3K: phosphatidylinositol 3-kinase; PSA: prostate-specific antigen; PTHrP: parathyroid hormone-related protein; RANKL: receptor activator of nuclear factor-jb ligand; SDF-1: stromal-derived factor-1; SFK: Src family kinases; TGF-b: transforming growth factor-b; TGFBR1: type I TGF-b receptor; TIMP: tissue inhibitor of metalloproteinases; upa: urokinase-type plasminogen activator; upar: upa receptor Grant sponsor: NIH/NCI; Grant numbers: T32 CA009666, P50 CA DOI: /ijc History: Received 22 Nov 2010; Accepted 28 Jan 2011; Online 1 Mar 2011 Correspondence to: Gary E. Gallick, Department of Genitourinary Medical Oncology, Unit , The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA, Tel.: , Fax: , ggallick@mdanderson.org organs such as the liver, lungs and brain, but it has an unusually high propensity for metastasizing to the bone. In one autopsy study, 80% of the men who had died from prostate cancer possessed bone metastases. 3 Most current treatments for individuals with bone metastases have only palliative effects, with little effect on long-term survival. 4,5 Thus, gaining a better understanding of the mechanisms by which prostate cancer metastasizes to the bone is critical to the development and use of therapies to improve survival of patients. In this review, we discuss the steps of prostate cancer metastasis and its growth within the bone. Events in Prostate Cancer Progression Development of Castrate-Resistant Disease Androgen receptor The dependence of prostate cancer cells on androgen stimulation was first described in a seminal article by Huggins and Hodges. 6 Androgen binds to the androgen receptor (AR) and translocates to the nucleus, where the binding of this complex to androgen responsive elements affects the transcription of androgen-regulated genes [e.g., prostate-specific antigen (PSA)] and ultimately stimulates proliferation and inhibits apoptosis of prostate cancer cells. Therefore, androgen-deprivation therapy by chemical and surgical castration has been the mainstay of the treatment for early metastatic prostate cancer. However, all patients invariably will progress at some point during the course of their disease as their tumor adapts to the androgen-deprived environment and becomes castrate-resistant. Several molecular mechanisms for the development of castration-resistant prostate cancer, an important step in progression of the disease to the bone, have been elucidated to date: Prostate cancer cells become hypersensitive to androgen stimulation by upregulation of AR expression. Also, autocrine and paracrine production of androgens is

2 2546 Prostate cancer metastasis to bone Table 1. Cell lines used in experiments described in this review Androgen receptor status Aspect of metastasis studied Cell line Origin (species) Bone lesion Method of studied Refs. ARCaP Human þ Orthotopic xenograft EMT 19 Intracardiac xenograft DU145 Human Invasion assay Invasion 20 LNCaP Human þ Mixed Migration assay Migration 21 LNCaP col Human þ Mixed Intratibial xenograft Adhesion 22 C4 2 Human þ Osteolytic Adhesion assay Adhesion 23 Intratibial xenograft Adhesion 24 Intratibial xenograft Bone remodeling 25 C4 2B4 Human þ Mixed Coculture, Migration assay Migration 26 Intratibial xenograft Bone remodeling 27 PC3 Human Osteolytic Coculture, Bone xenograft Invasion 28 Invasion assay Invasion 20 Bone xenograft Invasion 29 Binding assay Adhesion 30 Intracardiac xenograft Adhesion 31 Intratibial, Intracardiac xenograft Bone homing 32 Adhesion, Migration assay Extravasation 33 Intratibial xenograft Bone remodeling 25 PC3MM2 Human Osteolytic Migration assay Migration 34 Orthotopic xenograft Migration 34 PC3M-Pro4 Human Osteolytic Intratibial, Intracardiac xenograft Bone remodeling 35 P69 Human þ Adhesion assay Adhesion 23 MDA PCa 2b Human þ Osteoblastic Coculture, Intratibial xenograft Bone remodeling 36 LuCap 35 Human þ Osteoblastic Bone xenograft Bone remodeling 37 LuCaP 23.1 Human þ Osteoblastic Bone xenograft Bone remodeling 38 ACE-1 Dog Osteoblastic Intratibial, Intracardiac xenograft Bone remodeling 39 LAPC-9 Human þ Osteoblastic Intratibial xenograft Bone remodeling 40 upregulated in castrate-resistant prostate cancer. The AR may be activated in this setting by steroids other than androgens such as estrogens, and ligand-independent activation of the AR by receptor tyrosine kinases has also been described. Finally, in castrate-resistant prostate cancer, bypass pathways identified that contribute to AR-independent growth of prostate cancer cells, such as interleukin-6 (IL-6) signaling (described in more detail below). Several reviews have described the role of the AR in castration-resistant prostate cancer Interleukin-6 IL-6 is a glycoprotein implicated in progression to castrateresistant prostate cancer. 7 IL-6 is frequently expressed in prostate cancer cell lines, as early as benign hyperplasia, as well as in sera of patients with prostate cancer, in which expression increases in patients with metastatic disease. 14,15 The expression of IL-6 and its receptor has been consistently demonstrated in human prostate cancer cell lines. 11 IL-6 activates AR-mediated gene expression by activation of the AR through a STAT3 pathway in androgen-dependent LNCaP cells Overexpression of IL-6 increases PSA mrna in LNCaP cells (cell lines and variants discussed in this review are described in Table 1), partially ablating the requirement for androgen in growth of these cells. 41,42 Other signaling pathways, such as those mediated by Src and insulin-like growth factor (IGF) may also function to allow nongenomic signaling through AR after depletion of androgen, 7 and AR amplification is another common mechanism to permit AR signaling in a castrate-resistant environment. 43,44 Thus, many of the pathways discussed below not only contribute to classic steps in metastasis but also promote this process by constitutively activating AR. Details are described in several reviews. 44,45

3 Jin, Dayyani, and Gallick 2547 Events in Prostate Cancer Metastatic Process The classic model of metastasis of solid tumors, including prostate cancer, is guided by the seed and soil hypothesis first proposed by Stephen Paget in In Paget s model, the seeds (i.e., tumor cells) metastasize only to soil (i.e., specific organs) well suited ( fertile ) for the tumor s growth. Although this concept remains an excellent guiding principle, it does not entirely explain the molecular bases for organspecific metastases. Metastasis of prostate cancer, like that of other solid tumors, involves multiple steps, including angiogenesis, local migration, invasion, intravasation, circulation and extravasation of tumor cells and then angiogenesis and colonization in the new site. We will describe only the hallmarks of these events, which have been reviewed in extensive detail elsewhere We will then discuss our emerging understanding of properties of metastatic prostate tumor cells that facilitate their growth in the bone. Decreased cell adhesion and the epithelial to mesenchymal transition The initial stages of metastasis involve the detachment and migration of malignant cells from the primary tumor and their entry into the nearby blood or lymphatic vessels. In the normal prostate gland, epithelial cells have restricted migratory capability, in part because the basal cells inside the lumen attach to the basement membrane, forming a cell layer. 50 Normal epithelial cells above that basal cell layer adhere to each other as well as to the extracellular matrix (ECM). Cell-to-cell adhesion in normal epithelium is maintained by many different junctions, such as adherens and tight junctions, which are composed of protein complexes of cell adhesion molecules (CAMs), such as selectin and cadherin. Binding adjacent epithelial cells via cell junctions is critical for providing anchorage and communication between neighboring cells. During the process of malignant transformation, however, the adhesiveness of epithelial cells decreases. Specifically, early metastatic prostate cancer cells exhibit alterations in the expression of different molecules leading to decreased cell adhesion, such as E-cadherin. 50 This process is a major feature of epithelial to mesenchymal transition (EMT), which is now considered by most investigators to be critical in the development of a more migratory and invasive phenotype of epithelial tumor cells. 47,51 53 Experiments using prostate cancer cell lines in immunodeficient mouse models have confirmed that EMT is important in prostate cancer metastasis. For example, the epithelial-like cell line ARCaP E, which is derived from parental androgen-refractory ARCaP prostate cancer cells, was used for successive orthotopic inoculations into nude mice. 19 After only one inoculation, the ARCaP E cells from the resulting tumors exhibited EMT-like phenotypic changes. Cells from the tumors harboring those changes were then injected into a second group of mice, and those cells exhibited increased metastasis to several different organs, including the bone, demonstrating, at least in this model, a role for EMT in increasing metastatic potential. Figure 1. Src stimulation of migration and invasion through the E- cadherin/b-catenin complex. After engagement, activated Src phosphorylates E-cadherin/b-catenin complexes, with several results including recruitment of PI3K to this complex, activating downstream signaling pathways that lead to unbranching of actin filaments, eventual dissociation of b-catenin from the complex and functional loss of E-cadherin, all processes that promote tumor cell invasion. Src also promotes migration/invasion through focal adhesion kinase, as described in the text. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] E-cadherin and b-catenin. Specific molecular changes, many of them hallmarks of EMT, have been shown to play roles in adhesion, migration and invasion. E-cadherin and b-catenin are central in these processes. E-cadherin is a member of the cadherin family of CAMs in epithelial cells. 54 E-cadherin functions through the interaction of the calcium-dependent extracellular binding domain between adjacent cells, whereas its intracellular domain anchors the actin cytoskeleton through b-catenin, an adherence junction protein. 54,55 Thus, E-cadherin and b-catenin complexes have important functions in maintaining cell cell adhesions that connect the lateral side of epithelial cells. E-cadherin/b-catenin complexes are also affected by activation of the tyrosine kinase Src, 56 as shown in Figure 1. Other functions of Src in promoting invasion and metastasis are described below. One of the major features of cells undergoing EMT is cadherin switching, whereby, for example, E-cadherin (characteristically expressed in normal epithelial cells) is downregulated, and N-cadherin (characteristically expressed in mesenchymal cells) is upregulated. This cadherin switch occurs during prostate tumor progression. Tumor specimens from patients with higher grade prostate cancer (i.e., a Gleason score 8) had lower E-cadherin and higher N-cadherin expression than did those from patients with lower grade disease Decreased b-catenin expression was also associated with decreased expression of E-cadherin and also correlated with higher grade prostate cancer. 60 These clinical data are supported by studies from in vitro model systems that demonstrated that E-cadherin expression is reduced in more invasive human prostate cancer cell lines 61 and in invasive rat prostate tumors. 62

4 2548 Prostate cancer metastasis to bone Although these changes appear to be required for increasing metastatic potential, they may need to be at least partially reversed for outgrowth of tumors at the metastatic site. A recent study performed with clinical specimens showed reduced expression of E-cadherin and b-catenin in the primary prostate tumor but higher expression of both of those factors in metastatic prostate cancer cells in the bone. 63 These results suggest that mesenchymal to epithelial transition (MET) is required for the growth of metastatic prostate cancer cells once they reach the bone. An alternative possibility, as suggested by Saha, is that a subpopulation of migrating metastatic prostate cancer cells that overexpresses E-cadherin and b-catenin is responsible for cell adherence and subsequent growth in the bone. 63 Further work will be required to distinguish between these (and potentially other) possibilities. Integrins. The interaction between normal prostate epithelial cells and the ECM is critical for cell proliferation, survival and migration. 64 Integrins play multiple roles in cells including regulating focal adhesions, which are large macromolecular complexes that connect the cell s cytoskeleton to the ECM. Integrin-mediated signaling through focal adhesions promotes such diverse processes such as cell survival, motility and migration as well as crosstalk with growth factor receptors. Alterations in the expression of the integrins and molecules regulating the downstream signaling events they mediate are associated with prostate cancer metastasis. 65 The integrins consist of 24 glycoprotein heterodimers composed of combinations of 18 a and 8 b subunits that span the cell membrane and bind to specific subsets of ECM ligands. 66 In normal prostate epithelial cells, the predominant integrins are a5b1, a6b1 and a6b4, but avb3 and a3b1 integrins are also expressed. 50 Reduced or absent expression of a6 integrins in biopsy samples of prostate tumors correlates with the invasiveness of those cells. 67 In normal prostate epithelial cells, the a6b4 integrin forms hemidesmosomes that link the cytoskeleton to laminin-5 in the ECM. 68 In prostate cancer tissues, however, the expression of a6b4 integrin is frequently reduced, leading to the loss of hemidesmosomes weakening cell cell adhesion. 69 This change in integrin expression results in decreased adhesion of cells to the ECM, thereby promoting aberrant migration. 70 Signaling through focal adhesion kinase (FAK). Enzymes central to regulation of focal adhesions are the nonreceptor tyrosine kinases FAK and Src family kinases. FAK is a key regulator of focal adhesion turnover, and is critical to increasing cell migration. 71 During cell migration, focal adhesions are formed at the leading edge of the cell and used as an anchor on which a cell can pull itself over the ECM. Then, focal adhesions are disassembled at the rear edge of the cell when it moves forward and withdraws its rear edge. Blockage of focal adhesion turnover is known to increase the number and size of focal adhesions and inhibit cell migration. Alterations in integrin composition and function can thus affect migration through modulation of FAK function. In addition, overexpression of FAK itself is observed in high-grade and metastatic prostate cancer tissues compared to normal prostate tissues. 72 Increased levels of FAK in prostate tumor PC3 cells also correlates with increased metastatic potential. 73 Src family of kinases. Other key signaling molecules in focal adhesions (but not found exclusively in focal adhesions) include the Src family of nonreceptor protein tyrosine kinases. Phosphorylation of FAK after association with integrins leads to Src binding, followed by Src phosphorylation of FAK, recruiting signaling and nonsignaling molecules into focal adhesions. 74 Many recent studies have shown that aberrant activation of Src family of kinases (SFKs) by multiple mechanisms, including association with FAK, play important roles in prostate cancer progression. 75 SFKs may be activated by numerous mechanisms, including constitutive association with FAK as well as with growth factor receptors such as c- Met and insulin-like growth factor I receptor (IGF-IR) that are frequently overexpressed in prostate cancer. Src activation leads to rearrangement of the actin cytoskeleton and increased focal adhesion turnover and migration. 76,77 Constitutive Src activation (whether through association with FAK or other Src-binding partners such as aberrantly expressed growth factor receptors) is sufficient to activate numerous signaling pathways affecting proliferation, apoptosis, angiogenesis, cell cycle regulation and migration. Src/ FAK-mediated activation of the phosphatidylinositol 3-kinase (PI3K), Rho and Ras are important for tumor cell migration. Activation of the PI3K pathway by Src is known to increase cell survival. Activated Rho pathway is responsible for increasing cell motility, 78 Ras signaling is involved in increasing cell proliferation, angiogenesis and migration. 79 Specific mechanisms of Src activation have been detailed in many reviews. 76,80,81 In prostate cancer, Src is highly expressed in patient samples 82 and is associated with decreased patient survival and metastases. 83 Src activity is increased in castration-resistant prostate cancer, 83 and is higher in bone metastases than in primary tumors (Parikh and Gallick, unpublished). Aside from affecting focal adhesion turnover, Src activation promotes migration through many other molecules including interleukin 8 (IL-8), which is transcriptionally derepressed by activated Src phosphorylation of STAT3, leading to STAT3 binding to IL-8 promoter. 84 Inhibition of Src blocked IL-8 induced migration in prostate LNCaP cells. 21 Recent work has demonstrated that Src inhibition directly affects metastasis in nude mouse models. SFK kinase inhibitors decreased proliferation, invasion and migration in prostate cancer cells We showed that sirna knockdown of Src in metastatic prostate PC3MM2 cells inhibited migration in vitro, 34 and was sufficient to inhibit metastases to the lymph nodes in orthotopic nude mouse models. In contrast, knockdown of Lyn, another SFK, inhibited proliferation, consistent with previous reports, 88 suggesting that in prostate cancer, distinct SFKs play distinct roles in tumor progression and metastasis. Therefore, in mouse models, SFK activation

5 Jin, Dayyani, and Gallick 2549 directly contributes to prostate cancer metastasis. As discussed below, Src also affects osteoblast and osteoclast functions that contribute to the growth of prostate cancer in the bone. Clinical trials using Src inhibitors for patients with prostate cancer with bone metastases are discussed below. Prostate cancer invasion: Roles of proteases Prostate cancer invasion requires partial degradation of the ECM. The ECM is composed of basement membrane and connective tissue. Partial degradation of the ECM is an obligatory step in metastasis. The families of proteinases most associated with ECM degradation in prostate cancer are matrix metalloproteinases and serine proteinases such as urokinase-type plasminogen activator. Matrix metalloproteinases. Matrix metalloproteinases (MMPs) are a family of zinc-binding proenzymes, consisting of 24 different members. The proenzymes are inactive until proteolytic cleavage. ProMMP-2 is activated by membrane Type 1 MMP. 89 Once activated, MMP-2 (as well as MMP- 13) activates MMP In primary prostate tumor tissues, both the level of MMP-9, and the ratios of MMP-2/-9 to tissue inhibitor of metalloproteinases-1 (TIMP-1) are increased relative to normal prostate epithelium. These levels and ratios are further associated with high Gleason score and poorer patient survival Brehmer et al. 93 showed that loss of TIMP-1 was correlated with upregulation of MMPs in malignant human prostate cancer tissues. Further, in patients with metastatic disease, high concentrations of MMP-2 and MMP- 9 have been observed in plasma. 89 Thus, levels of MMP-2 and MMP-9 may be useful prognostic markers in prostate cancer. 94 However, a recent prostate tissue microarray study compared the level of MMPs and showed that only MMP-9 expression was prognostic. 95 This study did not look at secreted proteins, which may explain the differences in the conclusions with respect to MMPs as prognostic markers. In vitro studies have demonstrated that deregulation of a number of signaling pathways increase the expression of MMPs. 96 MMPs are negatively regulated by TIMPs. 96 Culturing minced explants of prostate tumor tissues in medium for 8 10 weeks, Lokeshwar et al. 97 demonstrated elevated levels of active MMP-2 and reduced levels of TIMP-1 in medium from prostate cancer tissues compared to that from benign prostate hyperplasia, suggesting that there is an imbalance of MMPs and TIMPs, i.e., upregulation of the former and downregulation of the latter, in prostate cancer. Other MMPs have different functions that contribute to prostate cancer metastasis. A recent study used a transgenic mouse model for prostate tumor formation and metastasis in mice in which the SV40 large T antigen is overexpressed only in prostatic neuroendocrine cells (Table 2). This mouse strain was then crossed with mice of the same genetic background in which MMP-2 was functionally deleted. 98 Regardless of MMP-2 expression (or lack thereof), prostate tumors developed. However, in the mice expressing the SV40 antigen in which Table 2. Transgenic mouse models described in this review Mouse Aspect of metastasis studied Refs. SV Tag TRAMP Invasion 98 SV40 Tag; MMP-2 / Invasion 98 SV40 Tag; MMP-7 / SV40 Tag; MMP-9 / MMP-2 was knocked out, reduced lung metastases and increased survival were observed. In contrast, using the same SV40 Large T antigen-expressing mice, functional deletion of MMP-7 or MMP-9 did not affect metastasis development, although blood vessel size was reduced. 98 Perhaps the most striking report related to roles of MMPs in prostate cancer bone metastasis comes from the study of MMP-12 in coculture systems. Nabha et al. 28 cocultured PC3 cells with bone marrow stromal (BMS) cells, and demonstrated increased production of MMP-12 in PC3 cells. These results demonstrate that the interaction of BMS cells and PC3 increases MMP-12 expression in the tumor cells. This result was further confirmed by an in vivo study that demonstrated MMP- 12 was primarily expressed in PC3 cells injected in human fetal bone xenograft as opposed to subcutaneous PC3 tumors. The authors further demonstrated that decreasing MMP-12 expression with an RNAi strategy reduced the invasiveness of PC3 cells by reducing degradation of Type I collagen in the bone, providing convincing evidence that MMP-12 participates in bone-tropic metastasis. 28 Urokinase-type plasminogen activator. Urokinase-type plasminogen activator (upa) is one of the major serine proteinases involved in facilitating ECM degradation, as well as activating other latent proteinases such as MMPs involved in this process. 48 One of the major functions of upa-upa receptor (upar) binding is to convert plasminogen to plasmin, a broad-spectrum serine protease involved in ECM degradation Studies on clinical prostate cancer specimens have demonstrated that high expression of upa and upar are both associated with higher tumor grades (Gleason score 8), increased invasion and the presence of lymph node metastases. 102 Festuccia et al. 20 demonstrated that the expression of upa in prostate PC3 and DU145 cells activates plasmin and leads to activation of MMP-9 and MMP-2, which can be inhibited by neutralizing antibodies to upa. Additional evidence of the importance of upa in prostate cancer was derived from experiments of Dong et al., 29 who injected PC3 in which upa expression was reduced by sirna cells into fetal bone, followed by implantation of these bones into immunodeficient mice. The authors observed reduced tumor burden and bone degradation relative to PC3 cells in which upa expression was not decreased. These results provide strong evidence that expression of tumor-derived upa plays an important role in prostate tumor growth in the bone.

6 2550 Prostate cancer metastasis to bone Prostate-specific antigen. PSA is one of the androgen-regulated genes. 103 Its product is a 240 amino acid glycoprotein and belongs to the family of kallikrein-like serine proteases. 104 In its catalytic domain, PSA has the His-Asp-Ser sequence, which is characteristic of serine proteases, and thus, implicates independent protease activity (i.e., not via plasmin) in protein degradation. 105 PSA is exclusively secreted by prostate epithelial cells and therefore the most widely used serum marker to diagnose early prostate cancer and also to monitor the course of the disease during and after treatment. PSA is most abundantly found in the seminal plasma, where it mediates the liquefaction of coagulated seminal plasma after ejaculation. 106 The degradation of fibronectin by PSA in the seminal plasma indicated a possible role for PSA as a protease in prostate cancer invasion since fibronection is also one of the main components of the ECM. Indeed, in in vitro assays, PSA was shown to degrade fibronectin, and its inhibition with specific antibodies resulted in a dose-dependent decrease of the invasion of PSA-producing LNCaP cells. 107 More work is needed to better define the role of PSA in prostate cancer progression. Homing of prostate cancer cells to the bone Endothelium attachment. Once cancer cells intravasate into circulation, they must survive in the circulation, attach to the vascular endothelium, and then extravasate into the bone. These later steps in metastasis are the most poorly understood, but likely require interaction of tumor cells with other cells for metastasis to develop. For example, prostate tumor cells bind to human bone marrow endothelial (HBME) cells with higher affinity than to other endothelial cells. 108,109 Although the detailed mechanism involving this preferential endothelial binding remains unknown, a dock and lock mechanism has been proposed. 110 One aspect of this model is that the endothelial cells in the bone constitutively express adhesion molecules such as P-selectin. 111 The sialyl-lewis X carbohydrate on prostate cancer cell surface molecules then associates with P-selectin through a low-affinity docking process. 112 The subsequent locking process of prostate cancer cells to endothelial cells is mediated by integrins, including avb3, a5b1 and a3b In a coculture model, the binding of PC3 cells to HBME cells was inhibited by integrin b1 antibody but not antibodies to other integrins, suggesting that attachment of PC3 cells to bone marrow endothelium is primarily mediated by integrin b1 in this model. 30 Other studies showed that human prostate epithelial P69 cells, immortalized with SV40 large T antigen (low tumorigenic potential), showed an increased number of cells bound to the HBME cell layer relative to C4-2 cells. However, as the levels of integrin expressions in C4-2 and LNCaP cells were similar, other factors besides integrins must be involved in attachment of prostate tumor cells to cells in the microenvironment (Fig. 2). 23 HBME cells also stimulate proliferation of PC3 cells in coculture models. 114 Therefore, bidirectional paracrine Figure 2. Homing and growth of prostate tumor cells in the bone. As described in the text, the specific mechanisms by which tumor cells home to the bone remain unknown, but are thought to occur through factors produced or expressed in both bone and tumor. Integrin b1 has been implicated in attachment of prostate cancer cells to the bone epithelium. Cadherin-11 (osteoblast-cadherin) is highly expressed in bone metastases and its knockdown decreases bone homing. Chemotactic factors such as SDF-1 released from the bone are implicated in bone homing of tumor cells. Once tumor cells reach the bone, the classic vicious cycle occurs, whereby factors from the tumor lead to bone destruction and bone formation, and factors from the bone released in this process promote tumor growth. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] interactions between prostate cancer cells and HBME cells are likely important in metastatic spreading of prostate cancer to the bone. 115 Colonization in the bone. Although some integrins discussed above are important in the early steps in metastasis, other integrins expressed in prostate cancer cells may be important in colonization of prostate tumor cells in the bone. Prostate cancer cells expressing integrins avb3 and a2b1, which are also expressed in osteoclasts, 116 facilitate tumor spreading in the bone. Integrin avb3 is expressed in metastatic PC3 cells and mediates migration on tissue culture plates coated with vitronectin and osteopontin, components of the bone In a mouse xenograft model, intratibial injection of C4-2 cells engineered to ectopically re-express integrin avb3 promotes tumor growth in the bone, whereas other inactive avb3 mutants did not. 24 Another integrin, a2b1, facilitates cell adhesion on collagen-i in the bone. The binding of metastatic prostate cancer cells to collagen can be inhibited by integrin a2b1 neutralizing antibodies. Intratibial injection of prostate cancer cells overexpressing integrin a2b1 into mice also promoted tumor growth in the bone. 22,120 Thus, expression of the osteoclast integrins avb3 and a2b1 by prostate tumor cells may help these cells attach and migrate to the bone matrix, facilitating outgrowth of metastatic tumor cells in the bone. Alterations in cadherin-11 may also be responsible, in part, for the bone-tropic nature of prostate cancer metastasis. Cadherin-11, also known as osteoblast-cadherin, is highly expressed in human prostatic bone metastases 31 as well as in

7 Jin, Dayyani, and Gallick 2551 prostate cancer cell lines derived from bone metastases, 121 but is not detectable in prostate cancer metastases to other organs. 31 Knockdown of cadherin-11 in metastatic prostate PC3 cells using an shrna strategy led to reduced incidence of metastasis to the bone after their intracardiac injection into immunodeficient mice. 31 Importantly, re-expression of cadherin-11 in C4-2B4 cells, a metastatic bone-tropic variant of LNCaP that lacks intrinsic cadherin-11 expression, increases cell migration and invasiveness as well as spreading and intercalation into osteoblast layer. 26 These results suggest that functions of cadherin-11 are bone specific and may facilitate the metastatic colonization of prostate cancer cells in the bone. Chemoattraction to the bone. Evidence from bone xenograft models has demonstrated that prostate cancer cells injected adjacent to the bone migrate toward bone. 122 Indeed, extracts from bone can promote chemotaxis and invasion of both PC3 and DU145 prostate tumor cells, whereas extracts from other organs have no such effect. 123 These data suggest that the bone contains chemotactic factors that attract prostate tumor cells. The expression of two such chemotactic factors, CXCR4 and stromal-derived factor-1 (SDF-1), are elevated in metastatic prostate cancer cell lines and in bone metastases Experiments support SDF-1 as a homing signal for metastatic prostate cancer cells to bone. Prostate cancer cell lines that localize to the bone express CXCR4 and also migrate across bone marrow endothelial monolayers toward a gradient of SDF-1 in vitro. 124,127 These observations are further supported by an in vivo study demonstrating that injection of neutralizing CXCR4 antibodies reduced bone metastases after intracardiac and intratibial injection of prostate PC3 cells into immunocompromised mice. 32 SDF-1 also increases PC3 cell adhesion to human umbilical vein endothelial cell monolayers and enhances trans-endothelial migration. 33 In contrast, other studies have provided evidence that CXCR4 expression alone is insufficient to promote bone metastases and requires other factors. When Hart et al. 128 used a CXCR4 antagonist peptide at maximal SDF-1 inhibiting concentration, invasion of prostate cancer cells on bone marrow endothelial barrier was not completely inhibited, suggesting that the CXCR4/SDF-1 axis is not the only chemotactic factor involved in bone metastasis. Other factors, such as epidermal growth factor, IGF and hepatocyte growth factor have also been shown to increase chemomigration or chemoinvasion of prostate cancer cells and are involved in chemoattractant mechanisms. 48 In addition to these molecules, the newly formed bone contains extracellular matrix proteins such as Type I collagen, osteonectin and bone sialoprotein, which also act as chemoattractants for prostate cancer cells migrating to bone. 129,130 Pathophysiology of Bone Metastasis Early explanations for the preferential bone metastasis of prostate cancer were that prostate tumors have specific phenotypes not found in other tumors that facilitate growth in the bone. 131 However, other cancers also metastasize to bone. 132 Thus, more recent studies have highlighted the importance of the bone microenvironment and the bidirectional interactions with prostate cancer cells. 115 The bone matrix is composed of 95% of Type I collagen and 5% of remaining noncollagen proteins and proteoglycans. 133 These noncollagen matrix proteins include osteopontin, bone sialoprotein and osteonectin, which are the preferred ECM molecules for attachment and growth by prostate cancer cells. 134,135 The cells in the bone marrow comprise not only osteoblasts and osteoclasts but also hematopoietic cells, adipocytes and immune cells. Together, the bone matrix and abundant growth factors secreted by these cells make the bone microenvironment a complex space and fertile soil for tumor growth. Current studies suggest that many of the processes that regulate bone cell maturation also promote prostate cancer bone metastasis. 136 Here, we discuss the role of cells in the bone microenvironment in facilitating prostate tumor growth in the bone and metastasis, with the understanding that the balance of growth factors and tumor microenvironment/tumor interactions contributes to either osteolytic or osteoblastic lesions. Osteoblasts and osteoclasts The bidirectional interactions of bone cells with prostate cancer cells suggest that not only can tumor-derived growth factors affect bone cells, but cells in the bone microenvironment also stimulate metastatic tumor growth. Numerous recent experiments have supported the importance of this interaction. One such study used primary mouse osteoblasts in coculture with MDA PCa 2b prostate tumor cells. Under these coculture conditions, MDA PCa 2b cell proliferation was stimulated, indicating the importance of paracrine interaction of the two cell types in promoting tumor growth in the bone. 36 Osteoblasts, derived from mesenchymal stem cells in the bone marrow stroma, are responsible for bone formation. 137 Osteoblasts become osteocytes when embedded in the bone or, alternatively, will undergo apoptosis if deposited to new bone matrix. 138 The newly formed bone matrix induced by osteoblasts will be hardened by mineralization with deposition of hydroxyapatite crystals to increase the resistance to compression. 133 The differentiation and growth of osteoblasts are primary regulated by complex signaling pathways including bone morphogenetic proteins (BMPs), IGFs, transforming growth factor-b (TGF-b) and Wnt. 139 Differentiated osteoblasts also secrete many of these growth factors, some of which are embedded in the bone matrix and can later be released by osteoclasts during bone resorption. 140 When metastatic cancer cells grow in the bone, they also produce many of these growth factors, resulting in stimulation of proliferation and maturation of osteoblasts and osteoclasts that, in turn, produce or release growth factors that further stimulate metastatic growth. The crosstalk between prostate cancer cells and osteoblasts/osteoclasts and other cells in the

8 2552 Prostate cancer metastasis to bone microenvironment of the bone is commonly termed the vicious cycle in which tumor growth affects bone modeling and bone modeling affects tumor growth (Fig. 2). Many signaling pathways contribute to the cycle. One example that has received considerable recent attention is signaling mediated through Src as discussed above. SFK/Abl inhibitors such as dasatinib inhibit tumor growth but also induce osteoblast differentiation and production of osteonectin, which promotes tumor cell migration and invasion. 141 Further, studies have demonstrated that Src activity is critical to osteoclast function. 142 Src / mice suffer from osteopetrosis due to lack of functional osteoclasts. 143 Phase 1 trials with the Src family kinase inhibitor saracatinib (or AZD0530) led to thickening of bones and decreasing in bone turnover markers. 144 Thus, Src inhibitors are in clinical trial for prostate cancer bone metastases both for their ability to affect tumor growth and the ability to interrupt the vicious cycle by affecting functions of numerous cells in the microenvironment. 145 Encouraging preliminary results from clinical trials using Src inhibitors in combination with chemotherapeutics agents emphasize the importance of targeting the microenvironment in which tumor cells reside as well as markers of osteoblast or osteoclast function as targets. Dasatinib is being used currently in a randomized Phase 3 trial in combination with docetaxel for men with metastatic castrate-resistant prostate cancer (ClinicalTrials.gov Identifier: NCT ). Results from a different Phase 2 trial with dasatinib alone showed decrease in osteolysis, 146 demonstrating that targeting of SFKs affects multiple pathways important to development of metastasis, and growth of metastatic tumor cells in the bone. However, the numerous and redundant mechanisms by which the microenvironment contributes to tumor growth in the bone emphasizes the need for a more complete understanding of the tumor bone microenvironment relationship. As described above, functions of osteoclasts are critical for releasing growth factors from bone matrix. Osteoclasts are derived from monocytes in the bone marrow. 133 The differentiation and maturation of osteoclasts is regulated primarily through the cytokines released by osteoblasts. Macrophage colony-stimulating factor-1 and receptor activator of nuclear factor-jb ligand (RANKL) promote fusion of monocytes to form multinucleated mature osteoclasts. 147 Mature osteoclasts bind to the surface of bone by avb3, avb5 and a2b1 integrins, followed by secretion of acid and lysosomal enzymes to degrade bone matrix. 133 Hence, bone resorption mediated by osteoclasts then releases several mitogenic growth factors from bone matrix, which is a key step for prostate cancer growth in the bone. 148 Bisphosphonates, a class of drugs with structural similarities to pyrophosphate, possess two phosphonate groups, and bind with high affinity to calcium which is abundantly found in the bone. Once ingested by osteoclasts, bisphosphonates induce apoptosis in these cells and thus prevent further bone loss. In patients with castration resistant prostate cancer, a group at high risk for skeletal complications, a large randomized trial showed that treatment with the nitrogen-containing bisphosphonate zoledronic acid resulted in a significant decrease in the number of skeletal-related events, which included bone fractures (vertebral or nonvertebral), spinal cord compression, surgery to bone, radiation therapy to bone (including the use of radioisotopes) or a change of antineoplastic therapy to treat bone pain. 149 Given the broad and somewhat subjective definition of skeletal-related events in this study, the use of zoledronic acid in this group of patients is not fully endorsed by all physicians. Bone remodeling Cancer metastasis in the bone almost invariably leads to an imbalance of bone formation and bone resorption, resulting in osteolytic or osteoblastic lesions. 139 Although the underlying mechanism for the imbalance remains unclear, different types of cancer cells have the propensity to secrete more osteoblastic or osteolytic factors. In prostate cancer, bone metastasis is usually osteoblastic with elevated bone formation, resulting in increased bone mineral density. 150 Osteoblastic metastasis of prostate cancer may be, in part, due to prostate cancer cells promoting osteoblast proliferation. The number of osteoblasts adjacent to prostate cancer cells is increased in bone metastases, whereas in osteoclastic tumors an increase in osteoclasts is observed. 151 Although bone formation is increased in prostate cancer bone metastases, the tumor-generated bone is abnormal, lacking typical lamellar structure of the normal bone and is thus termed woven bone. This type of bone formation easily leads to bone fractures that are frequently seen in patients with prostate cancer with bone metastases. However, the increased bone volume may help to confine tumor growth by limiting the space for cancer cells, which delays further progression of prostate cancer metastasis. 139 In contrast, bone resorption will increase in response to tumor-derived osteolytic factors, which is an important early step in initiating bone remodeling by creating more space for the tumor. Therefore, both osteoblasts and osteoclasts are highly activated, but dysregulated in the process of bone remodeling induced by growth of prostate tumor cells in the bone. Osteoblast-associated factors One mechanism by which prostate tumor cells induce osteoblastic lesions is by secreting osteoblastic factors, including endothelin-1 (ET-1), BMPs and IGF Here, we discuss several osteoblastic factors and their roles in stimulating osteoblast growth and contributing to metastatic prostate tumor growth in the bone. The molecular mechanisms contributing to osteoblastic lesions in other cancers have been reviewed elsewhere. 136,155 Endothelin-1. ET-1 is a small vasoconstricting peptide produced by the vascular endothelium, which has a key role in vascular homeostasis. ET-1 promotes bone formation by binding to ET receptor subtype A (ET A ), which is coupled to

9 Jin, Dayyani, and Gallick 2553 heterotrimer G proteins and activates secondary messenger systems to mobilize calcium and stimulate protein kinase C. This process stimulates phosphate transport, and is important for the initiation of bone matrix calcification. 156 ET-1 also increases osteoblast proliferation and inhibits osteoclast formation and motility The bone formation induced by ET-1 can be inhibited by the ET A atrasentan (pyrrolidine-3- carboxylic acid), in prostate cancer. 160,161 ET-1 may also increase osteoblast proliferation and bone formation by crosstalk with Wnt signaling, leading to the suppression of the inhibitor of Wnt signaling, Dickkopf-1 (DKK1). 162 Nelson and Carducci 160,161 first proposed that ET-1 is associated with osteoblastic metastases in prostate cancer, and found that exogenous ET-1 increases proliferation of prostate cancer cells. The level of plasma ET-1 is elevated in patients with osteoblastic prostate cancer metastases. 152 Atrasentan is a highly selective and potent ET receptor antagonist that significantly inhibits the development of osteoblastic response to cancer in bone in a variety of model systems. Several Phase 2 and 3 trials have evaluated its role in castrate-resistant prostate cancer. 163 To date, the clinical experience has shown that atresentan alone or in combination with docetaxel, a chemotherapeutic agent that is used to treat castrate-resistant prostate cancer, has modest activity in metastatic prostate cancer, and an ongoing Phase 3 trial is currently evaluating whether its addition to docetaxel in an earlier stage of disease might be of benefit to patients with prostate cancer (NCT ). Wnt signaling. Wnt is a soluble protein that binds to cellsurface receptors of the Frizzled family, which in turn activate members of the Dishevelled family proteins and subsequently stabilizes b-catenin, which then translocates to the nucleus and promotes multiple effects such as bone formation. Canonical Wnt signaling has been shown to stimulate osteoblast differentiation through b-catenin-induced gene transcription. 164 In prostate cancer bone metastasis, Wnt produced by prostate cancer cells stimulates osteoblast differentiation and, in addition, has autocrine effects on tumor proliferation. 165 Wnt signaling is also regulated by its antagonist, DKK1, which is primarily expressed in the early stages of prostate cancer, and is decreased in expression in bone metastases. 166 Inhibition of DKK1 in osteolytic PC3 cells stimulates their osteoblastic activity, whereas overexpression of DKK1 in prostate C4-2B cells changes a mixed osteolytic osteoblastic phenotype to an osteolytic phenotype. 27 These results suggest that DKK1 may be one of the molecular switches governing osteolytic metastases becoming osteoblastic in later stages of metastatic growth. In a recent study, overexpression of DKK-1 in prostate cancer Ace-1 cells, derived from a dog prostate carcinoma, increased subcutaneous tumor growth and the incidence of bone metastasis, but significantly decreased the osteoblastic phenotype of bone metastases in an intratibial mouse model. These results suggest DKK-1 has an inhibitory role in bone formation in prostate cancer-induced osteoblastic metastases via the Wnt canonical pathway. 39 Other studies suggest that noncanonical Wnt signaling also stimulates osteoblast differentiation, through BMP-dependent and BMP-independent signaling pathways. 167 Bone morphogenic proteins. Bone morphogenic proteins (BMPs) are members of the TGF-b superfamily 168 and are known to be involved in stimulating cancer cell migration. Osteoblast-derived BMP-2 can activate the Akt and ERK pathways, which in turn induce IKKa/b phosphorylation and NF-jB activation, resulting in the activation of b1 and b3 integrins and contributing to the migration of prostate cancer cells. 169 BMP signaling also activates the intracellular receptor Type I kinase, followed by phosphorylation of SMAD, which translocates to the nucleus and induces the expression of genes important for bone formation. 147 In clinical prostate cancer tissues, the expression level of BMP-7 was higher in osteoblastic bone lesions than in normal bone. 170 BMPs have been shown to be important factors for initiating osteoblast differentiation in vitro and in vivo. 171,172 Injection of an anti- BMP-6 antibody reduced osteoblast numbers and tumor growth of LuCaP 23.1 prostate cancer cells in implanted human bone in immunodeficient mice. 38 The osteoblastic effect of BMPs is further confirmed by expression of Noggin (an antagonist of BMPs) in LAPC-9 osteoblastic prostate cancer cells, which induced osteolytic bone metastases in an intratibial xenograft mouse model. 40 Further, ectopic expression of Noggin via a retroviral expression vector together with injection of RANK-Fc delayed the development of lesions induced by osteolytic PC3 cells. 173 These results suggest that BMPs play an important role in contributing to osteoblastic phenotype of bone metastasis in prostate cancer. A recent study suggests that BMP-4 signaling to induce apoptosis and Smad-mediated gene expression can be repressed by IGF-I through activating mtor signaling in prostate epithelial cells (NRP-152), suggesting a crosstalk between BMP and IGF signaling. 174 Insulin-like growth factors. Another important growth factor family affecting prostate cancer bone metastasis is the IGF family. IGF-I and II are abundant in the bone matrix and are released during bone resorption. 140 IGFs act through binding to the IGF receptors 1 and 2 (classic receptor protein tyrosine kinases) and promote cell proliferation, survival and angiogenesis. When aberrantly expressed, signaling through IGF- IR also promotes malignant transformation of fibroblasts NIH 3T3 cells. 175 IGFs promote osteoblasts to increase bone matrix apposition and decrease collagen degradation. 176 IGF- I is upregulated in prostate cancer metastases in the bone, and contributes to cancer cell proliferation and chemotaxis. 177,178 In clinical studies, levels of IGF also correlate with cancer progression, as high levels of IGF-I are associated with a Gleason score The protein level of IGFs and IGF-binding proteins (IGFBPs), which serve as carrier proteins for IGFs, could be mediated by proteolysis of IGFBPs.