Pro-osteolytic factors produced by prostate cancer cells
As noted, it appears that osteolysis is required before osteoblastic bone deposition in metastatic deposits. The cancer cells produce several pro-osteolytic factors that can enhance this initial bone resorption (Fig. 3).
RANKL stimulates osteoclast differentiation and action, and decreases apoptosis. OPG acts as a decoy receptor for RANK. The balance between OPG and RANKL is critical in controlling osteoclast activity. The prostate cancer cells produce factors that can stimulate or inhibit the activity and regulation of osteoclasts.
Prostate cancer cells produce RANKL and can directly initiate osteoclastogenesis and therefore stimulate bone resorption [16,18,23–25]. Up-regulation and expression of RANKL by prostate cancer cells and osteoblasts is controlled by several factors produced by the prostate cancer cells themselves, and therefore might act in a paracrine and/or autocrine fashion. These factors include PTH-related protein (PTHrP), IL-6 and IL-1, and PSA.
PTHrP is an endocrine hormone that evokes the same biological activity at the PTH receptor as PTH, increasing bone resorption. PTHrP is produced by prostate cancer cells and leads to the expression of RANKL on bone marrow stromal cells, inducing the formation of osteoclasts and bone resorption. This releases among other factors, TGF-β, which further increases PTHrP production by prostate cancer cells [4,24,26]. PTHrP might protect prostate cancer cells and osteoblasts from apoptosis, and act as a mitogen to promote tumour growth in addition to its osteoclastogenic properties .
Other potent osteoclastogenic factors produced by prostate cancer cells include IL-1 and IL-6 [16,18,26]. The latter is a potent stimulator of osteoclast formation and can enhance the effects of PTHrP on the formation of osteoclasts. Elevated serum levels of IL-6 correlate strongly with objective markers of prostate cancer morbidity, and suggest that it might be useful as a marker of prostate cancer activity and possibly disease progression [4,16,18,26]
Matrix metalloproteinases (MMPs) promote osteolysis and possibly metastasis by degrading bone matrix, and are secreted by prostate cancer cells (Fig. 4). MMP-2 and MMP-9 blood and urine levels are increased in patients with prostate metastases. MMPs are also active during osteoclast recruitment to sites of bone remodelling . The mechanism by which prostate cancer produces MMPs and induces bone resorption is not clear. It might involve induction of osteoclastogenesis, as inhibition of MMPs reduced the number of osteoclasts associated with prostate tumour growth in human bone implants in an experimental mouse model [18,27].
Figure 4. Summary of interactions between prostate cancer cells and bone marrow stroma and extracellular matrix (ECM). MAPK, mitogen-activated protein kinase; EMT, epithelial-to-mesenchymal transformation; TCF, T cell factor.
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Many of the effects of prostate cancer cells on the extracellular matrix pertain to metastasis and epithelial-to-mesenchymal transformation, the latter being necessary for the successful spread of cancer cells from the prostate to the bone metastatic site.
The kallikrein-related proteases, a family of serine proteases, have a specific involvement in both normal prostate and prostate cancer. PSA (or KLK3) is one such kallikrein-related protease. PSA hydrolyses the seminal vesicle proteins, seminogelin I and II, in ejaculate, liquefying the seminal clot . PSA has recently been shown to decrease OPG mRNA expression and increase RANKL mRNA expression, suggesting that PSA might induce osteoclast formation 
Pro-osteoblastic factors produced by prostate cancer cells
Many products of prostate cancer promote the hallmarks of the osteoblastic reaction, i.e. increased osteoid surface, osteoid volume and mineralization rate (Fig. 5). Studies suggest a positive association between the presence of metastatic prostate cancer and raised OPG levels. Whilst most OPG is likely to be produced by bone marrow cells, prostate cancer cells themselves have also been shown to produce OPG .
Figure 5. Details of interaction between prostate cancer cells and osteoblasts. CaP, prostate cancer; ET, endothelin; LEF, lymphoid enhancer-binding factor; UPA, urinary plasminogen activator; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; PDGF, platelet-derived growth factor.
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OPG in human prostate cancer cells has also been shown to be a survival factor due to its ability to bind a TNF-related apoptosis-inducing ligand (TRAIL) suppressing apoptosis. Production of OPG might therefore be a strategy for survival by providing a decoy target for TRAIL produced in and around tumour foci by patient monocytes and other cell types [24,26,30].
RUNX-2 controls transcription and when activated increases osteocalcin, bone sialoprotein, osteopontin, alkaline phosphatase and type I collagen. Protein kinase C is another intracellular messenger transduction system. Osterix is another transcription factor similar to RUNX-2.
BMPs and TGF-β are members of the TGF-β superfamily. BMPs have many functions in bone, including apoptosis, differentiation, proliferation and morphogenesis. Target genes of the BMPs in osteoblasts include OPG and RUNX-2 [18,24]. BMPs can induce uncommitted stem cells and myoblasts to express osteoblast characteristics, such as alkaline phosphatase or osteocalcin. Their osteogenic properties appear to be specific to the differentiation stage of target cells. They do not stimulate mature osteoblasts or fibroblasts to increase expression of these proteins [18,24]. Prostate carcinoma cells produce increasing levels of BMPs as they progress to a more aggressive phenotype, suggesting that up-regulation of BMP expression by cancer cells in bone is a critical component in the development of osteoblastic lesions [7,18,24].
TGF-β up-regulates RUNX-2 and similar controllers of osteogenesis. TGF-β levels are higher in patients with prostate cancer bone metastases than in those without [7,26]. TGF-β1 might act directly on the stroma, regulating angiogenesis and tumour progression as well as inducing differentiation in bone cell populations, inducing growth/survival factors by/for tumour cells, and regulating tumour cell attachment to matrices [16,26].
Serum levels of IGF-1 correlate with the risk of prostate cancer and the IGF-1 receptor is required for neoplastic transformation . Serum levels of IGF-BPs are inversely related to the risk of developing prostate cancer. IGF-1 binds receptors on osteoblasts activating RUNX-2 [7,18]. This might provide a link between IGF-1 and the development of osteoblastic metastases.
Endothelin-1 is a potent vasoconstrictor that belongs to a family of three 21-amino-acid peptides. The effects of endothelin-1 are mediated mainly through the endothelinA receptor. Endothelin-1 has been detected in osteocytes, osteoblasts, osteoclasts and vascular endothelial cells; it stimulates mitogenesis in osteoblasts which have both endothelinA and B receptors. It also enhances the effects of other osteoblast-stimulatory factors such as BMP-7 to induce bone formation [4,18,24,32].
Prostate epithelium produces endothelin-1 and has high-affinity receptors throughout the gland. Endothelin-1 levels are increased in patients with osteoblastic metastases from prostate cancer . Tumour-produced endothelin-1 might have paracrine (on bone cells) and/or autocrine effects (on tumour growth and apoptosis). Exogenous endothelin-1 increases prostate cancer cell proliferation and augments the effects of IGF-1, platelet-derived growth factor, EGF and FGF-2. It has also been shown that endothelin-1 production is increased by prostate cancer cells in contact with bone [7,18,24,32]. Substantial data associates endothelin-1 with osteoblastic metastases in prostate cancer.
VEGF is a key regulator of physiological and pathophysiological angiogenesis; it promotes endothelial cell proliferation, survival and migration. The effects of VEGF are mediated via several receptors. The two key receptor tyrosine kinases are VEGFR-1 and VEGFR-2. VEGF has previously been shown to regulate bone formation indirectly by controlling vascularity within the developing growth plate . VEGF has a direct effect on bone formation by stimulating migration and proliferation of human osteoblasts .
Prostate cancer cells produce VEGF, facilitating tumour growth by enhancing angiogenesis and possibly migratory ability. The VEGF produced by tumour cells binds neuropilin-1 on pre-osteoblasts, inducing osteoblast differentiation and, in conjunction with other tumour-related pro-osteoblastic factors, results in osteosclerotic lesions [7,33].
PSA and KLK2 mediate cell proliferation in both the normal and malignant prostate by interactions with the IGF axis. PSA has potent mitogenic activity for osteoblasts. This might be through elevation of IGF-1 acting as an osteoblastic growth factor increasing bone deposition. PSA might achieve this by degrading IGFBP-3, thereby increasing the bioavailability of IGF-1. Another pathway might be through PSA activating the latent form of TGF-β[4,26,28,29,34]. PSA might also have a direct role in modulating genes involved in bone remodelling, including up-regulation of genes such as RUNX-2, osteopontin and TGF-β.
PSA increases bone deposition by cleaving PTHrP ; the latter has many effects on the bone and prostate cancer cell populations, but its degradation might reduce bone resorption, thereby tending toward increased deposition [4,7,28,29,34,35].
Urinary plasminogen activator is another serine protease produced by prostate cancer that acts as an osteoblast growth factor. This might be due to increasing IGF-1 levels by hydrolysing IGFBP-3 and activating latent growth factors such as TGF-β similarly to PSA [4,7,18].
The Wnt pathway has been implicated in the development of osteoblastic metastases in prostate cancer in several ways. Wnt signalling by the prostate cancer cells might promote osteomimicry. Expression by tumour cells of BMP, osteopontin, the osteopontin receptor CD44 and RUNX-2, and the ability to produce mineralized matrix has been reported. The Wnt pathway might be involved in this osteomimicry, in that both osteopontin and CD44 are Wnt-regulated genes, and the canonical Wnts stimulate osteoblast mineralization and differentiation [8,9].
Other evidence indicating the involvement of the Wnt pathway is conflicting, showing the complexity of the Wnt pathway and its role in bone metastases. Hall et al. suggested an elegant mechanism of Wnt involvement in prostate cancer osteoblastic metastases. They proposed that the involvement of Wnt agonists and antagonists is integrated with many of the previously mentioned factors to produce a phasic model; Fig. 6 provides an overview of interactions between prostate cancer cells and bone.
Figure 6. Overview of the complex interactions between prostate cancer cells and the bone microenvironment promoting tumour establishment and growth of osteoblastic metastases. The shaded area illustrates metastatic cascade factors which promote prostate cancer cell metastases from the primary cancer deposit. ECM, extracellular matrix; CaP, prostate cancer; EMT, epithelial-to-mesenchymal transformation.
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In this integrated phasic model of metastatic prostate cancer, initially the prostate cancer cells target bone and establish metastases and produce pro-osteolytic factors such as RANKL, IL-6, PTHrP and the Wnt antagonist and inhibitor of osteoblastic activity DKK-1. The osteolytic activity releases growth factors stored in the bone, modifying the bone microenvironment, which then alters the prostate cancer phenotype. Tumour cells then produce osteoblastic factors including BMP, PTHrP (which can act as an anabolic factor by inhibiting osteoblastic apoptosis) and factors which block osteoclastic activity, such as OPG. DKK-1 expression also decreases, activating the Wnt pathway, which increases osteogenesis. This therefore transforms an initial osteolytic phenotype to an osteoblastic one. As the deposit expands, it outgrows its initial blood supply, producing hypoxia, inducing VEGF and endothelin-1 expression to promote angiogenesis. Both the cytokines also have osteoblastic activity, enhancing bone production . This model might explain why apparently conflicting data has been reported by various research groups, due to the phasic activity of the factors involved.