Pathogenesis of Bone Metastases: Role of Tumor-Related Proteins

INVESTIGATIONS ON the pathogenesis of cancer metastasis to bone have received increased attention by scientists worldwide. This is good news for patients that suffer from the problematic consequences of bone metastases from breast, prostate, thyroid, kidney, or lung cancer, but it also has highlighted the complexity of the disease process and the difficulties in studying the mechanisms of metastasis to bone in vivo and in vitro. Alterations in bone morphology and function caused by metastases are the result of sequential dysregulation of multiple genes, their protein products, and their respective biological functions in both cancer cells and normal cells that interact with the cancer. Knowledge of the pathobiology of the primary tumor, its progression with time to an invasive and metastatic phenotype, vascular spread to bone, entry into the bone microenvironment, growth in bone, and alteration of bone cell function and bone morphology will be required to understand the pathogenesis of bone metastases. This scope of knowledge necessitates the collaborative efforts of scientists with expertise in cancer and bone biology. It is important that we use our current understanding of cancer and bone pathophysiology to develop an accurate picture of the roles different genes and proteins play in the pathogenesis of bone metastases. There are significant challenges to studies of bone metastasis that may represent part of the reason why this field has received relatively limited attention in the past. These include the physical difficulty of manipulating bone as a tissue, the comparative difficulty of studying bone in vitro, the complexity of obtaining antemortem samples of bone metastases from human patients, and the relative lack of animal models that effectively mimic human disease. In all of these areas, great strides have been made, but much is yet to be accomplished. For example, it has been very difficult to identify animal models that mimic the osteoblastic metastases of prostate cancer in humans.1-3 In part, this is because of the fact that bone metastases are an uncommon phenomenon in animals with spontaneous cancer in contrast to humans.4 Mammary neoplasia is prevalent in rodents (mice and rats) and domestic dogs and cats. If these animals develop distant metastases it is usually to the lungs, and bone metastases are rare.5-7 In the absence of spontaneous animal models, experimental animal models have been developed, such as injection of tumor cells into the left ventricles of rodents or direct injection into bone.1, 8, 9 The definition of bone metastasis can vary depending on the endpoints examined, because the pathogenesis of bone metastasis by cancer is a multistep process. This is an important concept in correlating clinical and experimental data with the overall pathogenesis of bone metastasis. For example, bone metastasis can be defined as radiographic evidence of altered bone morphology using conventional X-ray radiology. This is an easily understood definition of metastasis but represents a late stage of disease progression and likely underestimates the extent of metastatic bone disease. Scintigraphy, computed tomography, magnetic resonance imaging, and histology will show significantly greater numbers of lesions.1, 10 An experimental procedure that results in reduced numbers of metastases solely based on radiological evidence can be interpreted incorrectly as reducing the actual incidence of bone metastasis. In contrast, bone metastasis may be overestimated by sensitive procedures, such as polymerase chain reaction (PCR), which can identify intravascular tumor cells or latent tumor cells in the bone marrow compartment that might not progress to form destructive bone lesions. Cancers that frequently metastasize to bone sequentially develop altered cellular characteristics during their progression to a metastatic phenotype.11, 12 Progression may involve inhibition of gene expression (such as tumor suppressor genes), activation of gene expression, or alteration in post-translational modifications of proteins. The sequential progression of tumor cells to the metastatic phenotype and some of the genes and proteins involved are summarized in Table 1. Not all cancers develop the phenotype necessary for metastasis to bone. Breast and prostate cancers have a high incidence (60–80%), thyroid, lung, and kidney cancers have a moderate incidence (30–50%), and gastrointestinal cancer has a low incidence (5%) of metastasis to bone.13 This suggests that different types of cancer have varied patterns of progression resulting in phenotypes that may or may not be permissive for metastasis to bone. Growth and differentiation of the primary neoplasm, controlled by growth factors, oncogenes, tumor suppressor genes, and genes that regulate differentiation or apoptosis, will affect the incidence of bone metastasis. The importance of gene expression in the primary tumor was exemplified by a recent investigation by Henderson et al., in which production of parathyroid hormone-related protein (PTHrP) by breast cancer was associated with a less malignant and less invasive phenotype with reduced incidence of bone metastasis.14 An early step in developing the metastatic phenotype is induction of local tissue and vascular invasion by cancer cells. This represents a transformation of the tumor cell from a relatively immobile cell to a more primitive motile cell that has fewer or modified cell-to-cell junctions, can penetrate the basement membrane, and migrates through connective tissue stroma. This process mimies early development of epithelial tissues in which migration of epithelial cells is necessary for organogenesis. Invasion is associated with altered expression of cell adhesion molecules and tissue inhibitors of matrix metalloproteinases and increased expression of proteolytic enzymes, such as the matrix metalloproteinases.11 The anatomical pattern of blood vessels can modify the pattern of cancer metastasis to bone. Most cancers are likely transported to bone by the arterial system after deposition into the venous circulation via the lymphatic and thoracic duct. An exception may be retrograde venous spread of prostate cancer to vertebrae via Batson's paravertebral venous plexus.15 Bone receives approximately 5–10% of the cardiac output, and bone metastases occur predominantly in areas of active red marrow.16, 17 This may be because of both the anatomical vascular pattern and the “seed and soil” concept of tumor growth (see below). Injection of cancer cells (that have a propensity to metastasize to bone) into the left ventricle of laboratory rodents results in metastases most commonly at the proximal and distal sides of the growth plate.18 Predisposition of tumor localization at these sites may be partially caused by the vascular anatomy of the growth plate, because growth plates remain open in adult laboratory rodents. There is an intricate arteriolar blood supply to the physis (especially in growing animals, such as laboratory rodents) with capillaries or sinusoids that complete a 180° turn at the growth plate. The turbulence or stasis of blood caused by this pattern may enhance tumor cell localization at this site. In addition, the sinusoids at the growth plate may have increased permeability or porosity at the junction of the primary spongiosa and mineralizing cartilage. This is also the site of predisposition to development of osteomyelitis caused by thromboembolic sepsis in growing animals and humans. In addition to anatomical pattern, adhesion of cancer cells to endothelial cells in bone also may be a requirement for entry into the bone marrow compartment. Adhesion due to integrins or other cell adhesion molecules may selectively enhance cancer cell localization in bone and even at specific sites in bone.19 It is reasonable to speculate that endothelial cell adhesion molecules could be different at the growth plate compared with other blood vessels in the bone marrow. After localization and adhesion of cancer cells to blood vessels in the bone marrow, the cells must penetrate the endothelium to enter the bone marrow compartment. This is facilitated by protease secretion by cancer cells, but chemoattraction also may play an important role in the migratory pattern of the cancer cells. Because metastases occur predominantly to red (hematopoietic) marrow, proteins from the marrow or bone cells or proteins released at sites of bone resorption, such as type I collagen, osteocalcin, transforming growth factor β (TGF-β), or insulin-like growth factors, may stimulate migration of the cancer cells into the marrow compartment.11 To develop into a definitive bone metastasis, the cancer cells that enter the bone marrow compartment must proliferate, induce angiogenesis, and ultimately alter bone structure. Inhibition of apoptosis also may play a role in tumor growth.20 Growth in the bone marrow represents a unique physical and paracrine interplay among the cancer cells and the bone microenvironment, including hematopoietic cells, bone marrow stroma, bone cells, and their matrices (seed and soil hypothesis).21 The seed and soil hypothesis is supported by the fact that certain cancers selectively localize in bone in spite of the fact that bone receives a minor fraction of the cardiac output. Most cancers (such as breast cancer) grow in the bone marrow compartment in close proximity to bone surfaces and stimulate osteoclastic bone resorption. Osteoclastic bone resorption is induced predominantly by soluble tumor products (such as PTHrP, interleukin-1, and tumor necrosis factor) that interact with bone cells in a paracrine manner.12 Proteins released from bone during osteolysis (such as TGF-β) may feed back on the tumor cells to further enhance the release of bone resorbing cytokines by cancer cells.8 In most areas of the bone marrow, cancer cells do not directly contact bone but are separated from bone by adjacent cancer cells, bone-lining cells, osteoblasts, or osteoclasts. In certain tumors, some cancer cells or tumorassociated macrophages adhere to the bone surface and participate in local osteolysis.22, 23 These forms of osteolysis may play a greater role in advanced lesions. In osteolytic lesions, osteoblastic bone formation may be inhibited by paracrine mediators, which can contribute to the reduction of bone at the metastatic site. Osteoblastic metastases are lesions having both increased bone formation and resorption.24 When bone formation is greater than resorption, the metastatic lesion is radiodense due to osteosclerosis. Osteoblastic metastases begin in the marrow cavity and are characterized by nonneoplastic woven bone proliferation on endosteal surfaces in association with cancer cells.1 Most prostate cancer metastases and few breast cancer metastases are osteoblastic. As the lesions progress in size there may be destruction of adjacent cortical bone. The tumor-related factors that induce new bone formation are under investigation and may include endothelin-1, prostate specific antigen, bone morphogenic proteins, urokinase, insulin-like growth factors, osteoprotegerin, or other substances.24-26 Osteoblastic metastases are rare in spontaneous animal cancers and very difficult to mimic in experimental animal models of bone metastases. The induction of mostly osteolytic bone metastases in laboratory rodents with human carcinoma lines is likely because of the progression of tumor lines away from the osteoblastic phenotype.1 It is important to evaluate carefully bone morphology at sites of bone metastases in rodent models. Osteolysis associated with tumor growth in the medullary cavity of bones in rodents often results in disruption of the cortex of the bones. Cortical lysis can cause a marked proliferation of periosteal new bone to stabilize the bone (form of callus). This is an expected physiological reaction to cortical lysis and does not represent an osteoblastic reaction, because proliferation of periosteal new bone is not caused by a paracrine action of the cancer cells. However, there have been recent successes in developing rodent models of true osteoblastic metastases (with intramedullary new bone formation) using certain human breast cancer lines injected in the left ventricle of nude mice, implantation of human prostate cancer lines in medullary cavities of nude or severe combined immunodeficient (SCID) mice, and intravenous injection of human prostate cancer cell lines in SCID mice with subcutaneous xenografts of human bone.2, 9, 29, 27 Certain cancers that metastasize to bone express proteins originally identified as bone-matrix proteins and include osteopontin, osteonectin, and bone sialoprotein (BSP).28, 29 The definitive role of these proteins in the pathogenesis of bone metastasis is uncertain, but they may enhance tumor growth, protection from immune surveillance, cell-to-cell adhesion, cell-to-bone adhesion, or migration into the bone microenvironment. Koeneman et al. have hypothesized that cancer cells that mimic bone cell function by secreting growth factors, enzymes, or matrix proteins that occur in bone could induce a phenotype that enhances bone metastasis.28 However, production of bone matrix proteins are not restricted to tumors that metastasize to bone.30, 31 Therefore, expression of these proteins does not necessarily confer a bone metastatic phenotype on cancer cells but may facilitate bone metastasis when combined with other tumor cell features. Conversely, these proteins may function differently in tumor cells that metastasize to bone, such as breast and prostate cells, possibly increasing their potential to metastasize to bone. Osteopontin is a calcium-binding phosphoprotein secreted by osteoblasts in bone and trophoblasts of the placenta. Osteopontin also is expressed by some osteotropic cancers, including breast and prostate cancer.32, 33 Osteopontin contains a conserved RGD (Arg-Gly-Asp) amino acid sequence and binds to integrins and the ubiquitous CD44 cell adhesion protein (and its variants).34, 35 Production and binding to the surface of cancer cells by osteopontin could enhance bone metastasis by facilitating cell-to-cell or cell-to-matrix adhesion, chemotaxis, and integrin-mediated signal transduction in bone. In addition, osteopontin can promote tumor cell survival by its ability to reduce macrophage nitric oxide-mediated cytotoxicity.36 Osteonectin is a multifunctional glycoprotein produced by osteoblasts and other cell types that plays a role in mineralization, cell-to-matrix adhesion, and angiogenesis. Osteonectin is produced by both prostate and breast cancer, in addition to other tumors including malignant melanomas and fibroblasts transformed with v-Ki-ras.37-39 Osteonectin promotes invasion and metastasis by enhancing matrix metalloproteinase activity in breast and prostate cancers.29 BSP is a secreted glycoprotein produced by osteoblasts, is present in bone matrix, and is produced by certain osteotropic cancers, such as breast, prostate, thyroid, and lung cancer.32, 40-43 In contrast, there is little BSP in normal prostate and breast tissue shown by immunohistochemistry.43, 44 BSP has an integrin-binding domain (RGD), which serves as a ligand for osteoclasts and can mediate migration, adhesion, and proliferation by binding to αvβ3 and αvβ5 integrins on breast cancer cells.45 RGD peptides have been used to block integrin-associated binding of thyroid cancer cells to bone matrix in vitro.46 It has been reported that human breast cancer cells synthesize BSP, which may promote mineralization of the tumors and render the carcinomas osteotropic.39, 41 Exogenous BSP peptides with the RGD sequence inhibited human breast cancer cell binding to extracellular bone matrix in vitro.47 BSP, secreted by BSP-producing cancers, can be measured in the serum.48 Most circulating BSP is protein bound and factor H (a component of complement) has been discovered as one of its binding proteins.49 Disruption of the BSP-factor H complex is necessary to measure total circulating concentrations of BSP. Serum BSP concentration may serve as a marker for osteotropic cancers. In this issue of the Journal, Waltregny et al. have investigated the presence of BSP in primary cancers of the prostate and breast and their metastases in visceral organs and bone using immunohistochemistry on banked tissue samples from autopsies of 15 humans. This is a timely investigation because there is a relative lack of studies on the pathogenesis of bone metastasis using human tissue samples because of the infrequency of biopsies or autopsy samples from patients with bone metastases. All of the primary breast tumors that were available for examination (5 of 8 and the bone metastases from the 8 patients were for BSP. Most of the primary prostate cancers (5 of and bone metastases (5 of were for BSP. These data of BSP expression by human breast and prostate cancers and the importance of the role of BSP in bone bone cells endosteal including osteoblasts and also for BSP. BSP was present in visceral metastases from multiple organs of the The of the in the primary and metastatic tumors was and were used to the primary tumors, bone and visceral metastases. There was in the primary tumors and their bone the that tumor cells from bone metastases that were close to bone were for BSP. There was a yet significant in BSP in visceral metastases compared with the bone metastases. These data were used to that BSP production by breast and prostate cancers might play a role in the pathogenesis of bone metastases. of the data also are in of the fact that there was in the primary tumors and their bone metastases. It is that the bone the tumor cells to their production of BSP to the primary tumor or that the of visceral metastases a of BSP production by the tumor cells. If the bone was necessary for the tumor cells to their BSP expression as it is as the for BSP production in the primary The the hypothesis that production of BSP by tumor cells may facilitate bone metastasis by tumor cells to to bone matrix In this BSP serve as a on cancer cells and acid sites in bone matrix to its role in binding to bone. However, binding of tumor cells to bone may not be a necessary component for the development of bone metastases for the cells must localize in the of bone and to endothelial cells, penetrate the capillaries or and and in the bone microenvironment. Most tumor cells in bone metastases are not in contact with the bone but are present in the marrow and are separated from the bone surface by marrow cells or bone cells. This is in 1. The of bone metastases suggests that entry and proliferation in the marrow is the most important for development of metastases. In and the that prostate or breast cancer cells are in close to the bone surface and BSP. However, that most breast cancer cells in the bone marrow are not in contact with bone. In addition, the cells in close association to the bone matrix in and have a different morphology compared with the tumor cells. are or more and the are This could be caused by altered tumor cell morphology after binding to the bone surface or it could be because of BSP of bone-lining cells the cancer cells from the bone as shown in of metastatic tumor cells with (such as and (such as be to that cells in contact with bone are cancer cells and that there are osteoblasts or cells cancer cells from the bone immunohistochemistry was by et al. to show that metastatic human breast cancer cells directly adjacent to bone were associated with matrix metalloproteinase expression and bone the bone resorption induced by the breast cancer cells in bone surfaces with compared with resorption induced by Bone resorption can promote the development or growth of bone metastases by multiple mechanisms released from resorbing bone, such as can serve as or growth factors for cancer has been reported to be induce matrix metalloproteinase expression, induce adhesion to via and stimulate expression in cancer In addition, bone resorption of the bone metastasis through the bone marrow, into cortical bone, and of the bone. Most of the bone resorption associated with metastatic cancer is by osteoclasts and is by paracrine factors, such as and produced by the cancer of bone directly by cancer cells or macrophages also may contribute to bone resorption in advanced lesions, but this is likely by osteoclastic bone on human bone metastases can be by in some of the tumor and bone to the relative of and tumor bone resorption, bone and the of bone formation and resorption compared with normal bone, and the and type of bone formation that is at sites of bone Production of BSP by cancer cells could facilitate binding of the cells to bone matrix (see and bone by matrix metalloproteinases secreted by the cancer cells. The of BSP in breast and prostate cells in this was predominantly in the but this does not the that BSP also was to the surface of the cells. and have reported that BSP is bound to the protein factor of factor H to BSP on the surface of cancer cells could the of cancer cell binding to bone However, of tumor cells by BSP and factor H may tumor cell survival by immune and cell It is likely that direct binding of metastatic cancer cells to bone matrix is not necessary for development or progression of bone metastases. Most metastatic tumor cells are not in contact with bone but are separated from bone matrix by adjacent tumor cells, hematopoietic cells, bone-lining cells, osteoblasts, or osteoclasts. However, binding of certain tumor cells to bone matrix via cell surface proteins, such as BSP binding to acid or binding to type I of bone matrix, has the potential to facilitate tumor cell lysis of bone and the release of and growth factors for cancer cells from bone. bone resorption may not be required for development of bone metastasis because of the of osteoclastic bone resorption, at in the early formation of bone metastases. cells can bone matrix proteins, but their role in the pathogenesis of bone metastases and It is that production of bone matrix proteins by cancer cells could enhance development of bone metastases by mechanisms other than facilitating binding of tumor cells to bone mechanisms include inhibition of immune of tumor cells, growth cell-to-cell adhesion, or migration in the bone marrow microenvironment. The by Waltregny et al. has to our understanding of BSP produced by metastatic human cancer, but on the role of bone matrix proteins in bone metastasis using primary human tumors will remain because of the to modify selectively tumor cell function in vivo in humans. This the importance of developing animal models of bone metastasis that mimic human disease. This was supported by from the of I and from The and from the of of for their and