Understanding and targeting osteoclastic activity in prostate cancer bone metastases

Introduction

Bone is a common site for metastasis in a number of tumors, including prostate, breast, and lung cancer; however, this review will focus primarily on PCa bone metastases. Even though only 8–14% of men have clinically evident bone metastases at diagnosis, it has been suggested that 85–100% of patients dying from this disease will develop bone metastases, even if subclinical (1). Bone metastases are a significant concern for patients as they are typically painful and increase the risk of catastrophic pathologic fracture and other skeletal-related events (SREs). Patient care is further complicated as there is no cure for these lesions despite significant effort to understand the underlying biology and specifically target them (2). The development of bone metastases in older patients with advanced disease is of significant concern as this population presents with decreased bone mineral density (BMD), exacerbating the risks of SREs leading to decreased quality of life and possible morbidity. One of the main line treatments for PCa, androgen deprivation therapy, also leads to decreased BMD, and may be a cofactor in SRE development. Skeletal metastases are typically described as osteolytic, those that lead to bone resorption, or osteoblastic, those leading to enhanced bone formation. Many lesions consist of a mix of osteolytic and osteoblastic components. It is often difficult to determine the nature of a metastasis by radiography alone. Typically bone markers, such as alkaline phosphatase (ALP), osteocalcin (OC), and tartrate-resistant acid phosphatase (TRAP) 5b, reveal mixed messages concerning changes in the bone environment, and may speak to the plasticity associated with bone metastasis (3, 4). Even though PCa bone metastases have an osteoblastic phenotype, they are thought to have an underlying osteolytic component important in tumor establishment and progression as previously reviewed (2, 5). One of the tenets of normal bone remodeling is that there cannot be bone production without prior bone absorption. It is only when these pathways fall out of balance that pathologies occur, and tumors are one mechanism than act to disturb this balance for their own growth promotion. Therefore, therapeutic intervention is required to improve quality of life and enhance survivability.

In normal bone, osteoblastic cells typically regulate bone turnover and osteoclastogenic activity. However, in the PCa microenvironment, many of these functions are co-opted by the tumor through the production of pro-osteolytic effectors. Several factors have been described as playing a role in the tumor induced osteolytic microenvironment. One such factor is receptor activator of nuclear factor κB ligand (RANKL) and it is supportive of the osteoclastic environment. RANKL expression has been shown to be induced by cancer cells leading to induction of osteoclast activity and osteolysis (6). Osteoprotegerin (OPG) is a soluble decoy receptor for RANKL, and therefore inhibits osteoclastogenesis and diminishes osteolysis in the PCa microenvironment. Parathyroid hormone-related protein (PTHrP) has been implicated as having a growth promoting role on PCa once it metastasizes to the bone. Interleukin-6 (IL-6) may also increase osteoclast numbers by promoting the growth of pre-osteoclastic cells. Matrix metalloproteinases (MMPs) and cathepsins lead to the breakdown of the extracellular matrix allowing for degradation of mineralized bone by osteoclasts, and increased invasion and migration of tumor cells. Together, these factors help describe the role that osteoclasts play in the promoting PCa growth in the bone microenvironment, and provide plausible pathways for the inhibition of tumor growth in the bone.

Osteoclast Biology

Osteoclasts are the primary mediators of bone resorption. In orchestration with osteoblasts, the primary mediators of bone formation, and osteocytes, the conductors of these two cell types, bone homeostasis is typically maintained. Osteoclasts help maintain bone structure and function through continual remodeling of the microenvironment in response to a number of physical and chemical stimuli. Osteoclast growth and differentiation is controlled by a number of well described processes (Figure 1). The colony forming unit granulocyte- macrophage (CFU-GM) is part of the monocyte-macrophage lineage of cells, and the earliest precursor to osteoclasts (7, 8). The CFU-GM undergoes a relatively well described progression of differentiation that has been previously reviewed (8, 9). Briefly, macrophage colony stimulating factor (M-CSF; colony stimulating factor, CSF-1), and interleukins-1, -4, and -6 (IL-1, IL-4, and IL-6) expand the osteoclast precursor pool and promote the formation of bone marrow macrophages (Figure 1) (9, 10). At this point, RANKL and M-CSF lead to promotion of a mature osteoclast through the expression of numerous genes associated with a mature osteoclast phenotype. Activation of the osteolytic phenotype is promoted by the presence of RANKL, PTHrP, and IL-6 (Figure 1). These cytokines lead to subsequent bone resorption which occurs through a defined process which has been previously reviewed in great depth (9, 11, 12). After activation and bone resorption is completed, osteoclasts undergo apoptosis which is enhanced by the presence of TGF-β and may be exploited by therapeutic intervention (e.g. bisphosphonates) (13). PCa cells exacerbate this system by creating many of the pre-osteoclastic cytokines described above, and will be discussed in greater detail below.

Although a number of factors have been implicated in the differentiation of immature osteoclasts, it is well accepted that RANKL is required for terminal osteoclast differentiation and induction of osteolytic activity. RANKL is balanced by the antagonist OPG, which helps to regulate osteoclast activity, and thus bone resorption (14). Osteolysis begins when a mature osteoclast is activated by RANKL, leading to upregualtion of MMP-9 and osteoclast polarization. Osteoclast polarization leads to ruffled membrane formation and the osteoclast sealing itself to the bone. Subsequent release of acid, cathepsins, and MMPs lead to breakdown of inorganic and organic ECM and resorption of bone constituent proteins. PCa has been observed to promote bone resorption through the induction of RANKL producing an environment conducive to PCa growth (Figure 2) (15).

PCa is a predominantly osteoblastic disease. However, bone resorption precedes bone production, and thus it is understood that even osteoblastic processes have an underlying osteolytic component. Formation of a vicious cycle is based upon tumor growth promoting the release of calcium and growth factors stored in the bone and extracellular matrix (ECM) which lead to further promotion of tumor growth (2, 12). Indeed, it has recently been suggested that osteoblastic lesions occur at locations on trabecular bone upon which osteoclast resorption has recently occurred (1, 16, 17). This suggests that osteoclast activation may be a key early mediator preparing the metastatic niche and nurturing early PCa metastases. Akech et al. have shown that an induction of Runx2, a transcription factor typically involved in positively regulating osteoblast activity, may play a role in bone lysis (18–20). They found that increased Runx2 expression in PC3, a lytic PCa tumor, induced mediators of osteoclast activity whereas PCa cells with a mixed phenotype, osteoblastic and osteolytic components, such as LNCaP and its derivative C4-2B had lower levels of Runx2 expression.

One of the primary methods of treatment for PCa is castration leading to loss of androgen and inhibition of tumor growth. However, androgen deprivation therapy has also been associated with an increased risk of SRE and osteoporosis (21). These factors are compounded by the fact that older men already have decreased BMD due to andropause resultant from decreased sex steroid (testosterone) production (22, 23). The combined effects can be so great that they lead to bone loss greater than that of postmenopausal women (23, 24). Furthermore, alterations in calcium metabolism and parathyroid hormone lead to excessive bone resorption (25). Further coalescence of nutritional, genetic, and environmental factors accentuates osteoporosis (26). The combination of these factors makes the risk of SRE in men with advanced PCa a serious clinical dilemma. Current and novel therapeutics for PCa metastases must resolve possible conflicts with bone remodeling in aging individuals, so as not to exacerbate these conditions.

It has long been understood that physiologic bone remodeling begins with osteoclast activation. These processes may be mimicked by metastasizing PCa cells leading to underlying PCa pathology (8, 27–29). Recently, Cackowski et al. modulated fetal mouse osteoclast formation and showed that osteoclasts are necessary for angiogenesis, the growth of blood vessels, through an MMP-9 dependent mechanism (30). They further attributed these effects to the presence of RANKL, PTHrP, and MMP-9, as these factors are critical for regulation of osteolysis, but may be exploited by tumors to promote growth. It is therefore plausible that osteoclasts may promote angiogenesis in the tumor microenvironment, a critical ingredient to tumor growth (31–33). Similarly, PCa cells have been shown to induce an osteoclastic phenotype in mature peripheral blood mononuclear cells (PBMCs) by regulating CD11b, which may be related to a common hematopoietic precursor cell (34). Metastatic PCa cells in the bone microenvironment compete for locations occupied by mesenchymal stem cells which have been suggested to be occupying locations of recent bone resorption (35, 36). These observations detail the importance of osteoclasts not only in preparation of the pre-metastatic niche for tumor cells, but nurturing continued growth of the tumor once seeding is complete.

Mediators of Bone Resorption

RANKL

Even though there are a number of factors that lead to increased osteoclastogenesis, the primary mediator of osteoclast differentiation and promotion of the bone resorptive phenotype is RANKL. RANKL is a member of the tumor necrosis factor family, and is also known as tumor necrosis ligand super family member number 11 (TNFSF11) (69). RANKL has been shown to promote osteoclast differentiation, activation, adherence, and survival, leading to increased bone resorption (16, 70–75). Osteoclast activity under RANKL is important in the mediation of bone formation of cortical and cancellous bone, and has previously been shown to mediate bone volume, density, strength, and rigidity.

RANKL is known to exist in at least 3 active forms (RANKL1-3), two of which (RANKL1/2) are membrane bound (76, 77). The different isoforms have variant effects on the process of osteoclastogenesis (77). RANKL1 and RANKL2 contain transmembrane domains and are typically expressed on osteoblast membranes and thus require cell-cell interactions to activate osteoclasts (77). However, a soluble form of RANKL can be created by tumor necrosis factor converting enzyme (TACE) or membrane bound MMPs cleaving membranous RANKL (6, 78). Specifically, MMP-7 has been implicated in RANKL promotion in prostate cancer osteolysis (6). Evidence suggests that RANKL3, lacking the transmembrane domain, serves to bind RANKL1/2 and possibly inhibit osteoclastogenesis (76, 79).

Activity of RANKL is dependent upon binding to receptor activator of nuclear factor kappa B (RANK; TNFRSF11A), the primary receptor for RANKL (14). Activation of RANK in osteoclasts leads to direct activation of c-fos allowing it to translocate to the nucleus promoting the transcription of at least 24 identified osteoclastic genes, including NFAT-c1 and c-Src (8, 80–82). Activation of RANK polarizes the osteoclast allowing for bone resorption (8, 70). c-fos is critical for osteoclast formation, but is not required for multinucleation to occur (83, 84). These changes subsequently lead to promotion of osteoclastogenesis and osteolysis as described above. Constitutive activation of RANK through tandem repeat expansion is the primary mechanism driving familial expansile osteolysis (FEO) leading to focal lytic disease that can expand and lead to significant skeletally related events similar to those seen in PCa bone metastases (14). RANKL can also increase migration in PCa cells through mitogen activated protein (MAP) kinase p38 and c-Jun activation (85). RANK activation of TRAF5/6 leads to activation of JNK1, c-Src, MAPK, and NF-κB which have a number of roles, predominantly concered with cell growth, migration, and survival (9).

The role that RANKL plays is not isolated to that of osteoclasts, but some preliminary studies suggest that RANKL can also alter PCa phenotype directly (86). RANK has been shown to promote migration of PCa cells (85). PCa can also induce osteoclast indirectly by inducing osteoblasts to express RANKL on their membranes, thereby activating osteoclasts to resorb bone (86). Outside of the effects on cancer cells, RANKL also plays an important role on T-helper cells, and has been implicated in the immune inhibition of breast cancer metastases; it is plausible that a similar mechanism is at work in PCa with direct and indirect effects on osteoclastogenesis, though these mechanisms need to be further elucidated (87–90).

Numerous activators of RANKL promote bone resorption through the effects of RANKL. However, these factors are balanced through the presence of IL-4 and IL-13 which typically lead to the production of OPG, the primary antagonist to RANKL (91).

Osteoprotegerin (OPG)

Osteoprotegerin (TNFRSF11B), also known as osteoclastogenesis inhibitory factor (OCIF), is a 120 kDa homodimeric protein that acts as a decoy receptor for RANKL (14). Upon binding RANKL, OPG inhibits RANKL’s ability to activate RANK. This, in turn, diminishes osteoclastogenesis and osteoclast activation. Increasing the ratio of OPG to RANKL has been shown to result in increased bone mass (92). Knock-out of OPG in mice leads to severe osteoporosis due to lack of inhibition of RANKL (93). OPG is produced by maturing osteoblast cells to inhibit the maturation of osteoclasts. In bone, OPG is primarily located on the cellular surface of osteoblasts, but is also present on a variety of other tissues, such as lung, heart, and kidney. A single dose of OPG was found to inhibit more than 90% of osteoclasts within 48 hours in mice, showing the potency of OPG as an inhibitor of osteoclast function, but not necessarily osteoclast survival (71, 94). Inducers of OPG include tumor necrosis factor alpha (TNF-α) and bone morphogenetic protein 2 (BMP-2), both of which also play a role in osteoblast differentiation.

It is well described that induction of OPG leads to decreased bone loss under a number of scenarios (95). For example, Bateman et al. observed that hind-limb suspension, a method to investigate disuse and microgravity, mice could be treated with OPG and bone loss ameliorated (96). Furthermore, the production of OPG has been related to estrogen through competition with the estrogen receptor (97). The presence of the estrogen-response element binding protein is thought to be ubiquitous in bone, yet its role in males and PCa has yet to be defined, if it exists. Administration of OPG has been shown to prevent bone loss in ovariectomized mice, and significantly inhibits bone turnover (16). Due to the relation of Vitamin D3 and OPG, it has been observed that vitamin D deficiency leads to decreased PCa growth in bone of animal models (98). Furthermore, several studies suggest that OPG may be a reliable measurement of metastatic bone disease in PCa patients, possibly more so than PSA (99–101). However, a recent study by Corey et al. observed that C4–2B PCa cells overexpressing OPG protected the cells from apoptosis, yet limited growth in bone possibly due to increased BMD and trabecular bone formation (102). It is likely that decreased bone turnover and lysis physically restrict tumor growth. Furthermore, patients with advanced prostatic tumors with metastatic bone disease have been shown to have increased ratios of RANKL to OPG, suggesting that RANKL expression by the tumor may overwhelm endogenous OPG expression (3). It is well described that OPG is an integral component to bone homeostasis, and in conjunction with RANKL and RANK serves to mediate bone turnover. Even though therapeutics aim to mimic the effects of OPG in the metastatic setting further examination of the role of OPG in these patients is necessary to better understand the underlying biology (16).

Interleukin-6 (I L-6)

IL-6 is a member of the “interleukin 6-type cytokine” family and was first identified as a macrophage differentiation factor (103). Other members of this family include, but are not limited to: leukemia inhibitory factor (LIF), oncostatin M, ciliary neurotrophic factor, interleukin 11, and cardiotrophin 1 (47, 103, 104). Numerous physiologic functions besides osteoclastogenesis are associated with this family of cytokines, such as: antibody production from B-lymphocytes, thrombopoiesis, and hepatic acute phase reaction protein synthesis. IL-6 mediates its activities through the IL-6 receptor (IL-6R) which is composed of an 80 kD transmembrane portion (IL-6Rp80) that specifically binds a 130 kd membrane glycoprotein (gp130). The gp130 subunit is common to all members of the IL-6 family of cytokines and is important in signal transduction (105). Heterodimerization in the presence of IL-6 induces a signaling cascade primarily through, Janus activated protein kinases (JAKs), mitogen activated protein kinases (MAPKs), JNK, E2F/Rb, and STAT3 leading to activation of CREB, oncostatin M, and RANKL in osteoclasts (103, 106–110). In addition to the membrane bound receptor, a soluble form of the receptor (sIL-6R) can be produced by proteolytic cleavage or differential splicing of the 80 kDa subunit. sIL6-R can bind IL-6 and interact with gp130 to induce signal transduction and promote PCa growth (111). IL-6 has been found to be critical for osteoclast formation in vitro and in vivo through the suppression of NF-κ associated pathways (112, 113). Therefore, the production of IL-6 from PCa cells and its role in the bone microenvironment make it a critical cytokine to examine for bone metastasis.

Numerous studies have demonstrated that IL-6 can be correlated with progressive disease in PCa patients and may act as a growth factor for PCa cells (4, 114–116). It has further been shown that IL-6 correlates with tumor burden and PSA acting as a growth factor for PCa (117, 118). Men with castrate resistant PCa had a significant increase in IL-6 compared to men with localized disease, benign prostatic changes, and normal prostates (114, 115, 119). IL-6 and IL-6R have been shown to be expressed on normal prostate epithelium and in PCa (116, 120). It is hypothesized that expression of IL-6 and its receptor play a role in increasing proliferation, metastasis, and androgen independence in PCa by utilizing STAT3 as a signaling mediator (117, 120–122). Recently, Kim et al. have shown that inhibition of IL-6 in a metastatic PC-3 model led to inhibition of tumor growth and decreased metastasis (119). These effects appear to be tumor type dependent, as IL-6 can also inhibit the growth of melanoma, breast cancer, and osteosarcoma (103). IL-6 has been shown to be a critical mediator of multiple myeloma (MM). MM cells produce IL-6 to stimulate osteoclast leading to increased bone lysis which in turn allows for increased MM growth and invasion (31). Administration of anti-IL-6 antibodies has been shown to inhibit growth of subcutaneously injected PCa in animal models, and may have potential as a therapeutic for clinical disease (122, 123). These data describe the importance of IL-6 in PCa biology and metastasis.

PCa cells injected into the bones of mice have shown IL-6 upregulation, and it is believed that these changes can have direct effects on the localized bone microenvironment and bone remodeling (117). IL-6 promotes osteoclastogenesis primarily through the induction of osteoclastic precursors, and does not appear to induce RANKL directly (124, 125). Interestingly, these effects appear to be tightly related to the concentration of gp130 present on precursor cells. It has been proposed that the IL-6Rp80 itself is not able to induce signal transduction, but must interact with gp130. Therefore, it is likely that gp130 is the primary mediator of IL-6 activity in osteoclast precursor cells. The effects of IL-6 are believed to be independent of RANKL, but may help potentiate the effects of PTHrP.

Parathyroid Hormone Related Protein (PTHrP)

PTHrP is related to PTH, which is involved in regulating hematopoietic stem cell development and calcium regulation which it partially accomplishes through IL-6 (126, 127). Both molecules bind to the PTH-1 receptor (PTH-1R) due to similarities in their N-terminal amino acid sequences (128). PTH1R is a G-protein receptor that leads to PKA and PKC pathways activation (129, 130). PTHrP has been observed to be secreted directly from breast, lung, and PCa cells; all of which metastasize to bone. PTHrP was first identified due to its role in mediating hypercalcemia in tumor bearing patients (131). PTHrP typically acts in a localized paracrine or autocrine fashion. PTHrP leads to increased RANKL expression by osteoblasts, thereby indirectly leading to the promotion of osteoclastic activity (Figure 2) (9). It is plausible that PTHrP acts in concert with IL-6 to promote RANKL expression (132). However, PTHrP does not lead to increased 1,25-dihydroxyvitamin D production as does PTH, and thus does not control intestinal calcium resorption although it does play a significant role in renal calcium homeostasis. In fact, PTHrP is suppressed by 1,25-di-hydroxyvitamin D₃ by mediating post-transcriptional pathways, decreasing mRNA stability, and inducing protein degradation by PCa (133). It has also been shown that PTHrP can act on osteoblasts to increase IL-6 and LIF to activate osteoclasts and inhibit osteoblast differentiation (103, 134, 135).

In normal bone, PTHrP serves to help regulate bone development, specifically endochondral bone growth. For example, selective depletion of PTHrP from osteoblasts leads to decreased bone mass, inhibition of osteoblast survival, and trabecular osteopenia (136, 137). The ability of PCa to secrete PTHrP and the ability of this molecule to regulate bone absorption and serum calcium makes it an interesting target for PCa therapy. PTHrP has also been found to associate with PCa progression, especially bone metastasis (129, 138). Indeed, PTHrP secretion from the tumor microenvironment can lead to an increase in monocyte chemoattractant protein-1 (MCP-1), inducing osteoclast recruitment and differentiation while promoting a pro-tumorigenic immune infiltrate of monocytes (139–141). However, the role of PTHrP is relatively complex, as it may also act as an inducer of anabolic bone growth dependent on the differentiation state of effector cells (129). A recent paper by Liao et al. described overexpression of PTHrP from PCa cells lead to an increase in bone density (142). However, an increase in osteoclastogenesis was also observed. These observations along with those of others describe complex interactions dependent on the amount of PTHrP produced, the cells present in the microenvironment, and the differentiation state of all players in the PCa microenvironment (20, 139, 143–147).

Therapy

Conclusion

Prostate cancer skeletal metastases often have a lytic component underlying their osteoblastic phenotype. Several mechanisms are important in promotion of osteoclast development, and activation of osteolytic pathways leading to bone resorption. IL-6 has been shown to be an important mediator in differentiation of osteoclasts while mediation of the balance between RANKL and OPG is important in regulating bone resorption and osteoclast activity. It is important to continue investigation concerning the mechanisms by which prostate cancer cells illicit bone osteolysis and activation of osteoclasts for therapeutic gain. Much information concerning the interactions between PCa and osteoclasts is required. For example, many of the typical laboratory models fail to replicate the osteoblastic environment, yet many of them have strong osteolytic tendencies when analyzed in vitro and in vivo. Examination concerning the nature of these cell lines is important, and may yield greater context to the underlying pathobiology of PCa as is reflected in the osteolytic bone microenvironment of metastases. Our understanding of PCa biology and the role that osteoclasts play can help define novel targets. Novel therapeutics aimed at presently known targets, and those now under development should yield important insight into the inhibition of PCa growth. For example, the role that PTHrP plays in regulating RANKL expression in osteoblasts as a subsequent regulator of osteoclasts is relatively difficult and poorly understood. Increased description of these pathways may yield useful therapeutic developments for patients with advanced disease. Current clinical trials seek to define the role that novel therapeutics play in the inhibition of PCa bone lysis to improve patient quality of life and increase survival. Continued investigation into the biology of the metastatic prostate tumor bone microenvironment will enable increased target definition, therapeutic development, and inhibition of the pain associated with these tumors.