REVIEW ARTICLE

MECHANISMS OF DISEASE

Mechanisms of Bone Metastasis

G. David Roodman, M.D., Ph.D. N Engl J Med 2004; 350:1655-1664April 15, 2004DOI: 10.1056/NEJMra030831

Bone metastases are a frequent complication of cancer, occurring in up to 70 percent of patients with advanced breast or prostate cancer1 and in approximately 15 to 30 percent of patients with carcinoma of the lung, colon, stomach, bladder, uterus, rectum, thyroid, or kidney. The exact incidence of bone metastasis is unknown, but it is estimated that 350,000 people die with bone metastases annually in the United States.2 Furthermore, once tumors metastasize to bone, they are usually incurable: only 20 percent of patients with breast cancer are still alive five years after the discovery of bone metastasis. 3 The consequences of bone metastasis are often devastating. Osteolytic metastases can cause severe pain, pathologic fractures, life-threatening hypercalcemia, spinal cord compression, and other nervecompression syndromes. Patients with osteoblastic metastases have bone pain and pathologic fractures because of the poor quality of bone produced by the osteoblasts. For all these reasons, bone metastasis is a serious and costly complication of cancer.

TYPES OF BONE METASTASIS

Metastases have been characterized as osteolytic or osteoblastic (Figure 1FIGURE 1
Osteoclasts and

Osteoblasts in Normal Bone and Bone Metastasis.). This classification actually represents two extremes of a

continuum in which dysregulation of the normal bone remodeling process occurs (Figure 2FIGURE 2
Regulation of Bone Resorption (Panel A) and Bone Formation (Panel B). ). Patients can have both osteolytic

and osteoblastic metastasis or mixed lesions containing both elements. Most patients with breast cancer have predominantly osteolytic lesions, although at least 15 to 20 percent of them have predominantly osteoblastic lesions.4 In addition, secondary formation of bone occurs in response to bone destruction. This reactive process makes it possible to detect osteolytic lesions by means of bone scanning, which identifies sites of active bone formation. Only in multiple myeloma do purely lytic bone lesions develop. In contrast, the lesions in prostate cancer are predominantly osteoblastic.5 However, there is also increased bone resorption in the osteoblastic lesions of prostate cancer, and agents that block bone resorption can decrease bone pain and the risk of pathologic fractures.6

BONE AS A PREFERRED SITE OF METASTASIS

Several factors account for the frequency of bone metastasis. Blood flow is high in areas of red marrow,7 accounting for the predilection of metastases for those sites. Furthermore, tumor cells produce adhesive molecules that bind them to marrow stromal cells and bone matrix. These adhesive interactions cause the tumor cells to increase the production of angiogenic factors and bone-resorbing factors that further enhance tumor growth in bone.8 Bone is also a large repository for immobilized growth factors, including transforming growth factor , insulin-like growth factors I and II, fibroblast growth factors, plateletderived growth factors, bone morphogenetic proteins, and calcium.9 These growth factors, which are released and activated during bone resorption,10 provide fertile ground in which tumor cells can grow. This

seed-and-soil hypothesis of the mechanism of bone metastasis was first advanced by Ste phan Paget in 188911and is supported by findings in animal models of bone metastasis.

CONTROL OF NORMAL BONE REMODELING

The adult skeleton continually turns over and remodels itself through the coordinated activity of osteoclasts and osteoblasts on trabecular surfaces and the haversian system. In normal bone, there is a balanced remodeling sequence: first, osteoclasts resorb bone, and then osteoblasts form bone at the same site. Osteoclasts Osteoclasts arise from precursor cells in the monocytemacrophage lineage12 that differentiate into inactive osteoclasts. Activated osteoclasts resorb bone and eventually undergo apoptosis. As shown in Figure 2A, both locally produced cytokines and systemic hormones regulate the formation and activity of osteoclasts. The bone microenvironment plays a critical role in the formation of osteoclasts through the production of macrophage colony-stimulating factor and receptor activator of nuclear factor-B (RANK) ligand (RANKL)13,14 by stromal cells or osteoblasts. RANKL, a member of the family of tumor necrosis factors, is expressed on the surface of osteoblasts and stromal cells and is released by activated T cells.13 Most osteotropic factors, such as parathyroid hormone, 1,25-dihydroxyvitamin D3, and prostaglandins, induce the formation of osteoclasts by increasing the expression of RANKL on marrow stromal cells and osteoblasts rather than by acting directly on osteoclast precursors15,16 (Figure 3FIGURE 3
Receptor Activator of Nuclear Factor-B Ligand (RANKL) and

Osteoclast Formation.). RANKL binds the RANK receptor on osteoclast precursors and induces the

formation of osteoclasts by signaling through the nuclear factor-B and Jun N-terminal kinase pathways. A soluble form of RANKL produced by activated T cells has been detected in the joint fluid of animals with adjuvant arthritis.17 The importance of RANKL in the formation of osteoclasts has been demonstrated clearly by the technique of homologous recombination in which the RANKL or RANK gene has been deleted in mice (knockout mice). These animals lack osteoclasts, and as a result, severe osteopetrosis develops.18,19 In addition, the development of B cells and T cells is defective in these animals. A decoy receptor for RANKL, osteoprotegerin, is normally present in the bone marrow. 20 Osteoprotegerin, a member of the superfamily of tumor necrosis factor receptors, inhibits the differentiation and resorption of osteoclasts in vitro and in vivo. The ratio of RANKL to osteoprotegerin regulates the formation and activity of osteoclasts. Overproduction of osteoprotegerin in transgenic mice causes severe osteopetrosis, whereas the absence of osteoprotegerin results in marked osteopenia.21,22 The importance of RANKL in bone destruction has led to the development of recombinant osteoprotegerin and antibodies against RANKL as potential treatments for bone metastasis. Osteoclasts resorb bone by secreting proteases that dissolve the matrix and producing acid that releases bone mineral into the extracellular space under the ruffled border of the plasma membrane of osteoclasts, which faces bone and is the resorbing organelle of the cell.23 The adherence of osteoclasts to the bone surface is critical for the bone resorptive process, since agents that interfere with osteoclast attachment block bone resorption.24 Agents that affect the adherence of osteoclasts to bone or inhibit proteases

produced by osteoclasts, such as cathepsin K, are under development and may be useful for treating bone metastases. Osteoblasts Osteoblasts are the bone-forming cells. They arise from mesenchymal stem cells, which form osteoblasts, adipocytes, and muscle cells.25 A transcription factor that is critical for the differentiation of osteoblasts is Runx-2, or core-binding factor 1 (CBFA1). CBFA1 drives the expression of most genes associated with osteoblast differentiation.26 Bone does not develop in mice that lack the CBFA1 gene.27,28 The differentiation of osteoblasts is less well understood than the differentiation of osteoclasts. It is clear that there is an early osteoblast precursor that produces alkaline phosphatase and a more differentiated precursor that produces increasing amounts of osteocalcin and calcified matrix.29 Osteoblasts eventually become osteocytes. Bone morphometric proteins are critical factors that stimulate the growth and differentiation of osteoblasts.30 As shown in Figure 2B, many factors can enhance the growth and differentiation of osteoblasts, including platelet-derived growth factor, fibroblast growth factor, and transforming growth factor .31

OSTEOLYTIC METASTASIS
In osteolytic metastases, the destruction of bone is mediated by osteoclasts rather than tumor cells.32,33 However, the factors responsible for the activation of osteoclasts vary depending on the tumor. In multiple myeloma, osteoclasts accumulate only at bone-resorbing surfaces adjacent to myeloma cells; their levels are not increased in areas uninvolved with tumor.34 In addition to the increase in bone resorption, bone formation is suppressed so that bone lesions in patients with myeloma become purely lytic.35 Several osteoclastogenic factors have been implicated in the increased activity of osteoclasts in myeloma.36 The leading candidates are interleukin-1, interleukin-6, macrophage inflammatory protein 1, and RANKL. Interleukin-1 is a potent stimulant of osteoclast formation,37 but levels of interleukin-1 produced by myeloma cells are extremely low.38 Sati et al.39 and Soutar et al.40 did not detect significantly increased levels of interleukin-1 in myeloma samples, suggesting that interleukin-1 may not be a major mediator of myeloma bone disease. Interleukin-6 is a growth factor or at least blocks apoptosis of myeloma cells.41 It is present in marrow plasma samples from patients with myeloma.42 Interleukin-6 is a potent stimulator of osteoclast formation43 and can enhance the effects of parathyroid hormonerelated peptide on the formation of osteoclasts in vivo.44 Interleukin-6 levels in the marrow have not consistently been correlated with the presence of bone lesions, however.45,46 When myeloma cells adhere to marrow stromal cells, the production of interleukin-6 by marrow stromal cells increases.47 Thus, interleukin-6 appears to have an important role in enhancing the growth or prolonging the survival of myeloma cells, but its role in myeloma bone disease remains to be determined. RANKL is a major mediator of myeloma bone disease. Several studies have suggested that myeloma cells produce RANKL,48,49 but it is unclear whether the amount of RANKL produced by myeloma cells is sufficient to induce the formation of osteoclasts. Instead, it may simply prevent apoptosis of osteoclasts. RANKL is produced by marrow stromal cells in myeloma. In the microenvironment of bone in myeloma, RANKL production is increased and osteoprotegerin is markedly decreased. 50 Blocking the binding of RANKL to the RANK receptor with a soluble form of the RANK receptor or osteoprotegerin inhibits bone

destruction in a mouse model of myeloma.51,52All these data suggest that RANKL is a major mediator of myeloma bone disease. Macrophage inflammatory protein 1 also appears to be a key regulator of bone destruction in myeloma.53,54 Macrophage inflammatory protein 1 is a potent inducer of osteoclast formation in vitro, independently of RANKL, and enhances both RANKL-stimulated and interleukin-6stimulated osteoclast formation.55 In approximately 70 percent of patients, myeloma cells produce macrophage inflammatory protein 1, and the level of this protein is elevated in bone marrow plasma. 53 Macrophage inflammatory protein 1 levels correlate strongly with the presence of osteolytic lesions; moreover, DNA microanalysis of myeloma cells has shown that the expression of the macrophage inflammatory protein 1 gene is markedly increased and correlates with bone disease.56 Furthermore, blocking expression of the gene for macrophage inflammatory protein 1 or the activity of macrophage inflammatory protein 1 in murine models of myeloma decreases both bone destruction and myeloma tumor burden.57,58 Macrophage inflammatory protein 1 also enhances adhesive interactions between myeloma cells and stromal cells by up-regulating the expression of 1 integrins on myeloma cells.57 Adhesive interactions between marrow stromal cells and myeloma cells increase the production of interleukin-6, RANKL, and macrophage inflammatory protein 1, further increasing bone destruction.

OSTEOBLAST DYSFUNCTION IN MYELOMA

Bone lesions in myeloma are purely lytic there is no osteoblastic response. This phenomenon explains the clinical observation that in about half the cases of myeloma, bone scans are normal in the presence of severe osteolytic bone destruction.59 The basis of the decreased osteoblastic response in myeloma is unknown. Myeloma cells can produce tumor necrosis factor , which inhibits osteoblastic growth and differentiation.60 However, tumor necrosis factor has not been implicated in the suppression of bone formation in myeloma. Although cocultures of two interleukin-6dependent myeloma cell lines with osteoblast-like human osteosarcoma cells reduced the amounts of osteocalcin produced by the cells, 61 the identity of the factor or factors responsible is unknown. Recently, Tian and coworkers,62 using gene-microarray analysis and immunohistochemical analysis, found that myeloma cells expressed dickkopf 1 (DKK1), a Wnt-signaling antagonist, and that the presence of high levels of DKK1 correlated with focal bone lesions in patients with myeloma. They further demonstrated that bone marrow serum from these patients that contained more than 12 ng of DKK1 per milliliter inhibited osteoblastic differentiation in a murine myoblast cell line. These data suggest that DKK1 may be involved in the inhibition of osteoblast differentiation in myeloma. It is likely that more than one factor is involved in the suppression of osteoblast activity in myeloma; this situation is analogous to the multiplicity of factors that increase osteoclast activity.

OSTEOLYTIC METASTASIS FROM BREAST CANCER THE VICIOUS CIRCLE

Tumor cells in breast cancer produce factors that directly or indirectly induce the formation of osteoclasts. In turn, bone resorption by osteoclasts releases growth factors from the bone matrix that stimulate tumor growth and bone destruction.63 This reciprocal interaction between breast-cancer cells and the bone microenvironment results in a vicious circle that increases both bone destruction and the tumor burden (Figure 4FIGURE 4
The Vicious Circle of Osteolytic Metastasis.).

Bone is an abundant source of inactive growth factors, which are activated during the bone-resorptive process10 and which can then stimulate the growth of breast-cancer cells. Parathyroid hormonerelated peptide is probably the factor produced by breast-cancer cells and most solid tumors that stimulates the formation of osteoclasts.64,65 Both parathyroid hormonerelated peptide and parathyroid hormone bind the same receptor (PTHR1) and induce the expression of RANKL on marrow stromal cells. Parathyroid hormone is the main peptide regulator of calcium homeostasis, and parathyroid hormonerelated peptide has biologic effects on bone similar to those of parathyroid hormone.66 In the amino acid sequences of parathyroid hormone and parathyroid hormonerelated peptide, 8 of the first 13 amino acids are identical, and both peptides have similar three-dimensional structures.66 The production of parathyroid hormonerelated peptide is increased in metastases of breast cancer to bone. Only 50 percent of primary breast cancers express parathyroid hormonerelated peptide, whereas 92 percent of metastases of breast cancer to bone produce the peptide.67 However, it is unclear whether this difference results from induction of the peptide in the bone microenvironment or whether tumors that produce the peptide are more likely to metastasize to bone. When breast-cancer cells from patients are injected into nude mice and metastasize to bone, they increase the production of parathyroid hormone related peptide.64 The peptide induces the formation of osteoclasts and bone resorption, which releases transforming growth factor . Transforming growth factor , in turn, further increases production of the peptide by the breast-cancer cells.68 An antibody against parathyroid hormonerelated peptide is being evaluated in patients with bone metastases from breast cancer. In the vicious circle of breast-cancer metastases (Figure 4), bone destruction increases local calcium levels, which promotes tumor growth and the production of parathyroid hormonerelated peptide.69 Breastcancer cells also produce, or induce, interleukin-6, prostaglandin E2, macrophage colony-stimulating factor, interleukin-1, and tumor necrosis factor ,70,71 which may also play an important role in the induction of osteoclast formation by breast-cancer metastases. Prostaglandin E2 can increase the expression of RANKL and directly enhance the effects of RANKL on the formation of osteoclasts. 71 Together, these data suggest that parathyroid hormonerelated peptide is a major mediator of osteolytic bone destruction by breast cancer and other solid tumors.

THERAPEUTIC IMPLICATIONS OF RESORPTION AND TUMOR GROWTH

There is a close relationship between bone destruction and tumor growth. For example, treating myeloma in mice with agents that block bone resorption but have no direct effect on tumor growth not only inhibits the formation of osteoclasts but also decreases the tumor burden.51,52 However, definitive evidence that decreasing bone destruction decreases the tumor burden in patients with bone metastasis is lacking. Gordon and coworkers72 reported that a single infusion of a potent bisphosphonate that blocks bone resorption markedly increased apoptosis of myeloma cells in vivo in 10 of 16 patients with newly diagnosed myeloma. In a large, randomized, double-blind, placebo-controlled study73 of the bisphosphonate clodronate, Powles et al. found that administration of the drug was associated with a decrease in both the incidence of bone metastasis and the death rate in patients with breast cancer who were at high risk for bone metastasis. However, blocking bone destruction does not appear to affect the growth of tumors in soft tissues,73 indicating the unique characteristics of tumor growth in bone.

OSTEOBLASTIC METASTASIS ANOTHER VICIOUS CIRCLE?

The mechanisms of osteoblastic metastasis and the factors involved are unknown. Endothelin-1 has been implicated in osteoblastic metastasis from breast cancer.74 It stimulates the formation of bone and the proliferation of osteoblasts in bone organ cultures,75 and serum endothelin-1 levels are increased in patients with osteoblastic metastasis from prostate cancer.76 Furthermore, in an animal model of osteoblastic metastasis, treatment with a selective endothelin-1Areceptor antagonist decreased both osteoblastic metastasis and the tumor burden.74 The antagonist had no effect on the growth of the tumor at orthotopic sites. These results suggest that blocking osteoblast-inducing activity by tumors may decrease tumor growth and osteoblast activity and suggest that a vicious circle may also be involved in osteoblastic metastasis in which tumors induce osteoblast activity and thus the subsequent release from the osteoblasts of growth factors that increase tumor growth. In addition to endothelin-1, platelet-derived growth factor,77 a polypeptide produced by osteoblasts in the bone microenvironment, urokinase,78,79 and prostate-specific antigen (PSA)80may also be involved.

OSTEOBLASTIC METASTASIS IN PROSTATE CANCER

Overproduction of urokinase-type plasminogen activator (u-PA) by prostate-cancer cells increases bone metastasis,78 and cells transfected with an anti-sense DNA to u-PA had one third as many metastases as did cells transfected with an empty vector. Human PC3 prostate-cancer cells produce a factor that is homologous to u-PA.79 Prostate-cancer cells also release PSA, a kallikrein serine protease. PSA can cleave parathyroid hormonerelated peptide at the N-terminal, which could block tumor-induced bone resorption. It may also activate osteoblastic growth factors released in the bone microenvironment during the development of bone metastases, such as insulin-like growth factors I and II or transforming growth factor .80 These data suggest that a vicious circle may also be responsible for osteoblastic metastasis. Bone metastases in prostate cancer are predominantly osteoblastic, with increased numbers of irregular bone trabeculae.81 However, markers of bone resorption are also increased in metastatic prostate cancer, although there is usually no histologic evidence of increased numbers of osteoclasts. In prostate cancer, levels of bone-resorption markers are higher in patients with bone metastasis than in patients without bone metastasis and reflect the extent of bone metastasis more accurately than does the PSA level.82 Recent clinical trials have suggested that blocking osteoclastic bone resorption decreases related skeletal events in patients with prostate cancer.6 However, in a murine model of prostate cancer, the inhibition of osteoclast activity did not inhibit the development of osteoblastic metastasis. 83Thus, it is unclear whether bone destruction precedes the development of osteoblastic metastasis or is a consequence of the increased bone formation. Yi et al. have shown in an animal model of osteoblastic metastasis that an initial phase of bone destruction is followed by extensive formation of bone.77 Their data suggest that bone resorption precedes bone formation in the development of osteoblastic metastases and that osteoclast activation plays an important role in the development of osteoblastic metastases.

BIOCHEMICAL MARKERS OF BONE TURNOVER FOR THE DETECTION OF BONE METASTASIS

The value of biochemical markers of bone turnover as a means of monitoring patients with bone metastases is still under investigation. Levels of bone-specific alkaline phosphatase, osteocalcin, and type I procollagen C-propeptide in serum are indicators of osteoblast activity, whereas serum levels of C-terminal telopeptide of type I collagen and tartrate-resistant acid phosphatase and urinary levels of type I collagen

cross-linked N-telopeptides are markers of osteoclast activity. All these markers have been used to assess the response to therapy or for the detection of bone metastases,84-86 but results have been variable. Urinary type I collagen cross-linked N-telopeptides and C-terminal telopeptide of type I collagen appear to be the most useful.84 In summary, bone metastases are among the most debilitating problems in patients with cancer. The molecular mechanisms responsible for both osteolytic and osteoblastic metastases are just being identified. The use of gene arrays and proteomics and the availability of appropriate animal models of bone metastasis have permitted the identification of factors produced by the tumor cells themselves or by the microenvironment in response to the tumor that mediate the process of bone destruction. These types of studies should result in the development of therapeutic agents to treat and possibly prevent this devastating complication of cancer.