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From: Cancer Drug Discovery and Development Bone Metastasis: Experimental and Clinical Therapeutics Edited by: G. Singh and S. A. Rabbani © Humana Press Inc., Totowa, NJ
1. INTRODUCTION
Metastases rather than primary tumors are responsible for the lion’s share of the morbidity and mortality arising from malignant disease. Patients with advanced breast or prostate cancers usually develop bone metastases and usually harbor the bulk of their tumor burden in the bone at the time of death. Although it is difficult to quantify the prevalence of bone metastases, natural history and autopsy studies give an indication of the scope of the problem. In breast cancer, the skeleton is the most common site for metastasis and first distant relapse, and median survival following detection is more than 20 mo (1). At their presentation with prostate cancer, 8% of white American males and 14% of African-American males have bone metastases (2), whereas the mean incidence of bone metastasis in an analysis of autopsy studies of prostate cancer is 70% (3). It is clear, therefore, that bone metastases are both a common manifestation in patients with breast and prostate cancer and often a prolonged one. Given these considerations and the fact that bone metastases are not usually clinically silent, their importance in the context of the overall suffering and management associated with these malignancies is evident.
Clinically, the lesions behave somewhat differently in the two cancers, with pathological fractures being relatively more common in breast cancer and severe, often intractable pain a classic feature of metastases in patients with advanced prostate cancer (4). Spinal cord compression as a result of vertebral metastasis is perhaps the most devastating complication of skeletal involvement with hypercalcemia and leukoerythroblastic ane- mia being the commonest systemic sequelae.
Overview
Sadmeet Singh, BM , BS , FRCS , and Gurmit Singh, P h D
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2. HOW TUMOR CELLS GET TO THE BONE
The principles underlying the dissemination of a primary tumor and the develop- ment of metastasis involve a series of steps that are the same for bone metastases as for hematogenous spread to other sites (5). Tumor cells from the primary lesion must first invade into the normal surrounding tissue, which they accomplish by producing pro- teolytic enzymes. This gives the tumor cells access to the microvasculature of the surrounding tissue, but they also make use of the neovascularization that the primary tumour induces, a process termed “angiogenesis” (6). Cancer cells then have to sur- vive in the circulation and travel to distant sites where, once arrested, they have to cross from the vascular network back into normal tissue. In order for a metastasis to be established, the cells need to be able to survive in the new environment. In the case of bone metastases, tumor cells entering the sinusoidal vascular channels of the bone marrow cavity have to traverse the sinusoidal wall in order to invade the marrow stroma. Angiogenesis is then required for the cells to form a macroscopic tumor at the endosteal surface of the bone. It is well known that this sequence of events is ineffi- cient; a very small proportion of the tumour cells that enter the circulation eventually end up as clinically detectable metastases (7). Recent data from experimental models of metastasis suggest that the initial steps of the metastasis cascade are completed highly efficiently and that the inefficiency of the process is apparent from the point at which tumor cells extravasate from the circulation at secondary sites (8,9).
A “homing” mechanism for cancer cells to specific organs has recently been proposed in experiments with breast cancer cells. Investigators showed that lung and bone stromal cells secrete a chemokine, CXCL12, and that breast cancer cells express high levels of a specific receptor, CXCR4. Using neutralizing antibodies to the receptor, they were able to block pulmonary metastasis. There has long been a search for such mechanisms and this finding offers exciting prospects for targeting bone metastases (10).
3. THE BONE MICROENVIRONMENT:
OSTEOLYTIC AND OSTEOBLASTIC METASTASES
The relationship between the tumor cells and the host tissue is a major factor in their ability to develop into macroscopic metastases. This is often referred to as the “seed” (the tumor cells) and “soil” (the metastatic site) hypothesis of metastasis after Paget’s paper of 1889 discussing the distribution of metastases in breast cancer (11). The unravelling of the molecular mechanisms involved in the interaction between the host microenviron- ment and tumor cell is seen as a key to the development of new therapies to deal with metastases.
It has been traditional to consider bone metastases arising from breast cancer as
osteolytic and those from prostate cancer to be osteoblastic. This difference between the
two diseases is evident in the radiological appearances of the bone metastases, but the
classification is an oversimplification of the mechanisms involved. Here, the molecular
crosstalk between the tumor cells and the bone microenvironment play a large part
in determining the nature of the lesions. Osteolytic and osteoblastic lesions are two
extremes, with the metastases of breast and prostate cancers usually containing elements
of both in different proportions.
3.1. Parathyroid Hormone-Related Peptide
The damage caused to the skeleton by bony metastases is usually in excess of what would be expected by the presence and volume of the tumor cells alone, particularly in the clinically lytic lesions of breast cancer. The osteolysis that causes this damage is a result of the activation of osteoclasts, specialized bone-resorbing cells that are derived from monocytes. The principle mediator of this osteoclast activation is parathyroid hor- mone-related peptide (PTHrP) (12). PTHrP was initially discovered during the search for a peptide sharing amino-terminal homology with parathyroid hormone from tumors associated with hypercalcemia (13) PTHrP plays a central role in the hypercalcemia of malignant disease by acting on the parathyroid hormone receptor to cause increased renal absorption and bone release of calcium (14). It is worth emphasizing here that although bone destruction is an important etiological factor in the development of hypercalcemia in malignancy, the role of renal mechanisms has tended to be underestimated.
Parathyroid hormone-related peptide is undetectable in health, but it is present in most patients with breast cancer and its levels are higher in bone metastases than in either soft tissue secondaries or in primary tumors (15). Osteoclastic bone resorption resulting from bone-induced tumor cell overproduction of PTHrP causes the release of active growth factors. A “vicious cycle” of bone loss is then established because the growth factors lead to proliferation of tumor cells, which, in turn, continue to produce osteolysis-promoting PTHrP (16). The expression of PTHrP in the biopsies of bone metastases from patients with untreated prostate cancer has recently been shown in an immunohistochemical study
(17). This finding is consistent with our understanding that osteoclast activation occursin prostate cancer, although the role of PTHrP in the pathogenesis of skeletal disease in this malignancy requires further investigation.
3.2. The RANK-RANKL System and Bone Metastasis
Following from the discovery of the role of PTHrP in the etiology of osteolytic bone metastasis has been the identification and characterization of a new cytokine system capable of regulating the proliferation, differentiation, activation and apoptosis of osteo- clasts (18). This cytokine system is comprised of receptor activator of nuclear factor κβ ligand (RANKL), its receptor (RANK), and its “decoy receptor,” osteoprotegrin (OPG).
The exogenous administration of RANKL to normal mice has been shown to increase the number and activity of osteoclasts and promote the development of severe osteoporosis and lethal humoral hypercalcemia. Recently, it has been shown that PTHrP-producing breast cancer cells led to bone erosion and the expression of RANKL when injected into the periosteum of mice skulls, whereas non-PTHrP-producing cells did not lead to destructive bone lesions (19). OPG acts as a endogenous receptor antagonist able to neutralize the biological effects of RANKL. A variety of both in vitro and in vivo data have demonstrated the antiosteoclastic effects and bone-protective actions of OPG as recently reviewed by Hofbauer et al. (20).
3.3. Growth Factors
Interactions between members of a number of growth factor families secreted by
breast and prostate cancer tumors and the bone have been observed. Members of the
transforming growth factor-β (TGF-β) family have been implicated in the development
of bone metastases in breast and prostate cancers at many levels. They are powerful in vivo stimulators of new bone and their involvement in the development of osteoblastic lesions in prostate cancer has been shown by a number of investigators (20). Recent evidence also suggests that TGF-β is involved in PTHrP-induced osteolytic metastases
(21) and the RANKL system (22).Prostate cancers secrete large amounts of acidic and basic fibroblast growth factors (FGFs). These have been shown to stimulate bone formation in vivo and thus act as mediators for osteoblastic metastases (23). Endothelin-1 is another growth factor shown to be associated with the development of metastases in prostate cancer. It is found at increased levels in patients with prostate cancer, and bone metastases (24) and prostate cancer cells have been shown to stimulate osteoblast activity by secreting it (25).
4. MATRIX METALLOPROTEINASES AND BONE METASTASIS The matrix metalloproteinase (MMP) family comprises over 20 zinc-dependent pro- teases that, collectively, are able to degrade all of the components of the extracellular matrix. As experience has accumulated with MMPs, so has the discovery that their substrates include a wide variety of extracellular proteins such as other proteinases, proteinase inhibitors, chemotactic molecules, cell surface receptors, and adhesion mol- ecules (26). MMPs play a role in the physiological remodeling of bone: They are involved in osteoclast recruitment to sites of remodeling (27) and are among the enzymes that degrade the mineralized bone matrix (28). Increased MMP expression has been docu- mented in nearly all epithelial tumors. In prostate cancer, MMP-2 expression has been found to be a marker for tumor vs normal tissue and to be correlated with tumor grade (29) and the presence of metastasis (30). Similarly, a wide variety of MMPs have been impli- cated in the pathogenesis and metastasis of breast cancer (31). MMP activity seems to be particularly relevant in the development of bone metastases as a result of the abundance of the extracellular matrix and its resistance to degradation.
5. THERAPEUTIC APPROACHES
The above-discussed molecular mechanisms all present opportunities for therapeutic intervention. The successful use of bisphosphonates for metastatic bone disease is an example of application of the understanding of these mechanisms for therapeutic pur- poses. They and some of the approaches, which will hopefully yield benefit in the future, are discussed in the following subsections. A schematic diagram of some of the mecha- nisms involved, the sites, and the modes of intervention discussed is shown in Fig. 1.
5.1. Bisphosphonates
These drugs are already indicated for the treatment of metastatic bone pain in breast and prostate cancer as well as for the prevention of skeletal-related events in breast cancer. In addition, they are a highly effective therapy for the treatment of hypercalcemia in patients with all tumor types, in the presence or absence of bone metastases, and are used for the treatment/prevention of osteoporosis in women.
All bisphosphonates have a central P-C-P-containing structure, which is able to bind
to mineralized bone matrix, and a variable side chain that determines the potency, side
effects, and precise mechanism of action. The rationale for the use of bisphosphonates
in metastatic bone disease lies in their inhibitory action against osteoclasts enabling them to diminish bone resorption. Bisphosphonates bind avidly to exposed bone matrix around resorbing osteoclasts, reaching very high concentrations at these sites. Once released from the bone surface, they are taken up by osteoclasts, where they disrupt the biochemi- cal processes involved in bone resorption. Bisphosphonates also cause osteoclast apoptosis and recent data suggest that they might be directly apoptotic to tumor cells (32), although the molecular targets for these actions remain unknown.
The antiosteolytic action of these drugs and their proven efficacy in decreasing the burden of bone metastasis in breast cancer (33) has led to the evaluation of their use in the management of skeletal disease in prostate cancer. This is based on the realization that osteolysis occurs in bone lesions from prostate cancer, but it occurs in the background of an intense osteoblastic reaction (16,34). However, data have been unconvincing. The most encouraging results have recently been published in a randomized controlled clini- cal trial assessing the efficacy of zoledronic acid (the most powerful bisphosphonate currently used) in patients with hormone-refractory metastatic prostate cancer (35). Some of the end points measured (skeletal related events and time to first skeletal-related event) demonstrated significant benefit in favor of the treatment arm. A review of the results, however, did not recommend the routine use of the drug in patients with metastatic prostate cancer on the basis of the lack of demonstration of net benefit and probable cost implications (36).
Fig. 1. Schematic showing some of the steps involved in the development of bone metastases along with sites and modes of current and proposed intervention.
5.2. Other Approaches Targeting Osteolytic and Osteoblastic Factors The elucidation of some of the molecular mechanisms involved in bone metastasis discussed earlier have opened new avenues for targeting of therapies. The endogenous decoy receptor to RANK, OPG, has been shown to effectively inhibit osteolysis and bone metastasis burden in murine models (37). RANK-Fc is molecule that acts in the same way as OPG and, again, has shown very promising results in suppressing bone resorption in an animal model of tumor-induced osteolysis (38). PTHrP has also been used as a therapeutic target. Using antibodies to PTHrP in bone metastases from breast cancer cells implanted into nude mice, investigators were able to show abrogation of osteolytic lesions (12). It is thought that these strategies might be more effective than using bisphosphonates both decreasing bone resorption and treating hypercalcemia.
5.3. MMP Inhibitors
Inhibition of MMPs is conceptually a very good aim in the management of metastatic
disease. MMPs are produced largely by stromal tissue in response to tumor cells, so
therapy targeted here theoretically avoids the problem of tumor resistance. Tumor cells
do, however, utilize their own MMP production (39) in order to intravasate from their
Fig. 2. Examples of femurs harvested from mouse bone metastasis model. The left-hand panel shows a control animal in which no tumor cells were injected and no drug was given. In the middle panel is a femur in which the animal received breast cancer cells. There is widespread destruction of the bone with metastatic tumour shown by the outline. The third femur is from an animal injected with tumor cells and treated with doxycycline. There is a dramatic decrease in tumor burden compared with the untreated animal.primary site and extravasate at the site of metastasis (40). Therefore, therapy is potentially active at multiple levels; against primary tumor cells, at the site of metastasis, and in the stroma. Following successful in vitro and in vivo studies, a number of MMP inhibitors have been tested in clinical trials for a variety of tumors, although not specifically for bone metastasis. Unfortunately, the results of phase III trials have been disappointing with little evidence of clinical efficacy; indeed, two trials were terminated early because of poorer survival in the treatment arms (41).
Encouraging preclinical data have recently shown MMP inhibitors to be effective against bone metastases in animal models of breast and prostate cancer. One group has demonstrated that an MMP inhibitor can decrease bone tumor burden and increase sur- vival in a mouse model of metastatic breast cancer (42). Another (43) has provided in vivo confirmation of in vitro results from our laboratory (44) that prostate cancer cells produce MMPs when introduced to the bone environment. Using a model in which human prostate cancer cells are injected into human bone implanted within a mouse, they observed that an MMP inhibitor prevented bone degradation and reduced tumor cell proliferation. Both of these recent studies have highlighted and exploited the relationship of the skeleton with the prostate to show a beneficial effect of MMP inhibition in bone metastasis.
5.4. Tetracyclines and Bone Metastasis
The use of the tetracycline family of antibiotics for the treatment and prevention of bone metastasis from breast and prostate cancer is a particular research interest in our laboratory. Tetracyclines are broad-spectrum antibiotics indicated in a wide range of Gram-negative and Gram-positive infections. The finding that led to the interest in the use of tetracyclines in metastatic bone disease was the discovery that they inhibited connective tissue breakdown by a nonantimicrobial mechanisms (45). It has since been determined that the principle mechanism by which tetracyclines achieve this is MMP inhibition. This is thought to be a result of their ability to chelate zinc ions on which MMP activity is dependent (46). Tetracyclines also inhibit the production and activa- tion of MMP proenzymes—the latent form that MMPs are secreted before activation by endogenous proteases (47). Tetracyclines have also been shown to directly inhibit the proliferation of human tumor cell lines, including breast and prostate in vitro (48,49).
Chemically modified tetracyclines that possess MMP inhibition activity but do not have antibacterial activity have recently been developed and these have been shown to successfully decrease bone loss in models of osteoporosis (50). The use of tetracycline to prevent bone loss has been given further support by the finding that tetracyclines induce apoptosis in osteoclasts (51). The final, but perhaps most compelling, rationale for the use of tetracyclines in the treatment or prevention of bone metastasis is the high concentrations they achieve in bone. They are quickly cleared from the bloodstream but accumulate in the skeleton, where their maximum therapeutic benefit is proposed (48).
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