Publications
APS Bulletin • Volume 7, Number 2, March/April 1997
Innovations in Practice
Richard B. Patt, Department Editor
Mechanisms of Bone Metastasis
R.N. Rosier, MD PhD; D.G. Hicks, MD; L.A. Teot, MD; J.E. Puzas, PhD; R.J. O'Keefe, MD
Metastasis remains the dominant problem area in the clinical care of the cancer patient. Many common malignancies have a strong propensity to metastasize to bone, including breast, prostate, lung, kidney, and thyroid carcinomas. In particular, the incidence of bony involvement can exceed 90% in metastatic breast and prostate carcinomas. Thus, given the high and rising incidence of these cancers, chronic bone pain, pathologic fractures, and other complications such as hypercalcemia are all-too-common problems. Pathologic fractures secondary to metastatic carcinomas are among the most frequent tumor-related problems seen by the orthopedic surgeon. Chronic bone pain due to metastatic carcinoma also represents one of the most difficult treatment challenges and greatest impediments to quality of life in cancer patients.
Recent scientific developments have enhanced our understanding of mechanisms of cancer metastasis and suggest new opportunities for both suppression and prevention of metastatic disease. In addition, advances in knowledge of the molecular mechanisms that govern bone resorption and cancer cell adhesion in bone present further opportunities for novel therapeutic strategies. Clinical applications for such new approaches could ultimately contribute to a badly needed paradigm shift in the prevention and therapy of cancer metastasis.
Metastasis involves an intricate and complex sequence of events and is fundamental to the definition of malignancy. An extremely diverse spectrum of neoplastic diseases shares this one feature of metastatic capability. The long-standing theory of "seed and soil" holds that metastasis results from both biological properties of the malignant cell and conducive host tissue factors. An increasing body of current data supports this hypothesis. Metastasis involves a series of cellular properties that result in specific events, including (a) cell motility; (b) expression of matrix metalloproteinases (MMPs), which confer ability to degrade extracellular matrix components; (c) ability to cross basement membranes, gain access to vascular or lymphatic circulation, and egress in a remote organ site related to the motility and MMP expression; (d) endothelial adhesion mechanisms that facilitate distant vascular or lymphatic escape; (e) chemotaxis conferring target organ selectivity; (f) selective cell adhesion to specific extracellular matrices or cellular components through cell surface receptors such as integrins; (g) ability to induce angiogenesis to support metastatic tumor growth; (h) local invasiveness related to MMPs and other proteases, and possibly to cytotoxic effects on the host tissue; and (i) continued uncontrolled growth driven by a variety of molecular mechanisms, including locally secreted host and tumor cytokines. The complex array of requisite steps for the occurrence of this pathologic process suggests multiple potential points of therapeutic antimetastatic intervention. As an example, interference with MMP function to prevent basement membrane penetration is currently an exciting and very active area of basic research that may yield new suppressive treatments for metastasis. The importance of the appropriate "soil" is also increasingly evident. We have long known that certain cancers have a predilection for particular organ distributions of metastasis, such as the tendency to metastasize to bone mentioned above for breast and prostatic cancers. Indeed, different tumors tend to metastasize to specific bony sites, and metastasis to distal long bones or small bones of the extremities are extremely rare with most carcinomas. When such acral metastases occur, they are typically associated with lung carcinomas, suggesting some specificity in the metastatic process. Traditional explanations attribute this to anatomic factors such as vascular and lymphatic distribution, but recent evidence indicates that local tissue factors may be a strong determinant. A convincing model involves a rat osteosarcoma, which when injected into the venous circulation, reproducibly metastasizes to the lungs (Kuratsu, Uchida, & Araki, 1992). When pieces of lung were removed and ectopically explanted in the subcutaneous tissue, injected osteosarcoma cells metastasized not only to the lungs but also to the lung explants. Furthermore, when pulmonary alveolar cells were isolated from fragments of lung tissue and were injected into subcutaneous tissue, injected osteosarcoma cells again metastasized to the explanted cell areas. This model provides strong evidence in support of the critical role of local tissue factors as determinants of target tissue specificity in the metastatic process.
Matrix Metalloproteinases and Metastatis
Another important factor that influences metastasis is the secretion of MMPs, which are necessary for local tissue invasion as well as penetration of basement membranes for access to avenues of remote spread. MMP research has enhanced our understanding of these enzymes and how they participate in the malignant phenotype, and constitutes an active and rapidly expanding field that may reveal new approaches to interfere with or suppress metastasis. The MMPs comprise a large and growing family of metal-dependent proteases with varying substrate specificity and tissue expression. There are currently 14 family members, the best-characterized of which are listed in Table 1. There is considerable overlap among MMPs regarding extracellular matrix substrate specificity, and they are ubiquitous in their expression. An appealing aspect of these enzymes as targets for antimetastatic therapy is that they are generally expressed at relatively low levels in normal adult tissues, but are upregulated in pathologic processes such as injury and repair or neoplasms. MMPs are critical to basement membrane penetration, where collagen type IV and other extracellular matrix proteins must be degraded to allow entry to or egress from vessels or lymphatics. The MMPs that have been most frequently implicated in the metastatic phenotype are the gelatinases (MMP2 and 9), stromelysins (MMP3, 10, and 11), and interstitial collagenase (MMP1).
Table 1. Matrix Metalloproteinases | |||
| MMP | Molecular Weight | Name | Substrates |
|---|---|---|---|
| MMP1 | 52-57 kD | Interstitial collagenase | Collagens I, II, III, VII, VIII, X, proteoglycan |
| MMP2 | 72 kD | Gelatinase | Collagens I, IV, V, VII, X, XI, elastin |
| MMP3 | 55-60 kD | Stromelysin | roteoglycans, fibronectin, laminin, collagens IV, V, IX, X, elastin, procollagen |
| MMP7 | 28 kD | Matrilysin (PUMP1) | Fibronectin, laminin, collagen IV, gelatin |
| MMP8 | 75 kD | Neutrophil collagenase | Collagens I, II, III, VII, VIII, X, proteoglycans |
| MMP9 | 92 kD | Gelatinase B | Collagen IV, V, elastin, proteoglycans |
| MMP10 | 55-60 kD | Stomelysin 2 | Same as stromelysin 1 |
| MMP11 | 55-60 kD | Stomelysin 3 | Unknown |
| MMP12 | 53 kD | Elastase | Elastin |
All MMPs have two highly conserved zinc binding domains: a catalytic site and a structural site. In addition, there are two conserved calcium binding domains. Thus, molecules with chelating properties can potentially interfere with these metal binding sites and inhibit activity, providing one obvious therapeutic approach. The enzymes are all secreted in a proenzyme form, with the propeptide folded in a way that it blocks the catalytic zinc site and maintained in this inactive conformation by a so-called cysteine switch. Upon cleavage of the propeptide, the MMP becomes activated and can degrade substrate molecules. MMPs can cleave the proenzyme forms of each other, and can therefore become activated in a cascade-type fashion. MMP expression is also regulated at the transcriptional level by growth factors, cytokines, and intracellular signaling molecules, including TGFß, TNF-*, and cAMP (Callaghan, Lovis, Rammohan, Lu, & Pope, 1996; Mann et al., 1995; Overall, 1994; Tanaka et al., 1995).
Manipulation of these regulatory pathways affords another possible approach to metastatic suppression. All cells that secrete MMPs also secrete endogenous inhibitor proteins of these enzymes, called tissue inhibitors of metalloproteinases (TIMPs). These molecules bind stoichiometrically to MMPs, maintaining them in an inactive state. Thus, the balance between TIMP and MMP expression ultimately controls the amount of matrix degradative activity present. Overexpression of TIMP experimentally by transfection of malignant cell lines leads to inhibition of metastasis in animal models, while underexpression causes enhancement of metastasis (Montgomery, Mueller, Reisfeld, Taylor, & DeClerck, 1994; Watanabe et al., 1996). Three forms of TIMP have been identified, with TIMP1 and TIMP2 being the most prevalent forms and TIMP3, a matrix-bound form, being less well characterized (Apte, Olsen, & Murphy, 1995). MMP expression has been reported to correlate with metastasis and prognosis in patients with several different cancers, as has underexpression of TIMP (Baker et al., 1994; Naylor, Stamp, Davies, & Balkwill, 1994; Onisto et al., 1995).
One of the most promising areas of anticancer research is the pharmacologic manipulation of MMP activity. An appealing aspect of this approach is that a number of agents with significant MMP-inhibiting activity that have minimal toxicity currently exist. Examples include tetracyclines and hydroxamates, which function by chelating the active metal-binding domains of the MMPs. Tetracyclines have demonstrated beneficial clinical effects in periodontal disease and arthritis, presumably through suppression of MMPs involved in these pathologic conditions (Greenwald, Moak, Ramamurthy, & Golub, 1992; Rifkin, Vernillo, & Golub, 1993). In addition, tetracyclines have been reported to suppress metastasis in animal models (Masumori et al., 1994).
Batimastat (BB-94), a synthetic hydroxamate MMP inhibitor, has been reported to have a dramatic effect on ovarian and colon carcinomas in animal models, and has also been used with some apparent benefit in a human clinical trial with these tumors (Wang, Fu, Brown, Crimmin, & Hoffman, 1994). A number of new MMP inhibitors with varying specificity are currently under development, exploiting the critical nature of the catalytic metal-binding site. In addition, retinoids that are cAMP analogs have been demonstrated to downregulate MMPs at a transcriptional level, while prostaglandins may upregulate these genes in some cells. Thus, the use of phosphodiesterase inhibitors, retinoids, or prostaglandin inhibitors could provide additional pharmacologic methods of inhibition of MMP expression in malignant tumors, although these approaches need considerable further study.
Besides MMPs, several other metastatic markers or predictors have been identified. A protein called nm23 (nonmetastatic 23), which is negatively associated with metastasis, is a nucleotide diphosphate kinase, and is also homologous to a transcription factor called PuF. While controversial, several studies have demonstrated the expression of this marker and its inverse correlation with poor prognosis and metastasis. However, some studies have failed to demonstrate a correlation, and the significance of nm23 expression as a predictor remains uncertain. Other markers predictive of metastasis include maspins, metastasin, and several protooncogenes. Identification of novel metastatic markers may lead to an understanding of their functional role in the metastatic process and thereby to new therapeutic or preventive strategies.
Bone Metastatis
Metastasis to bone requires both progressive displacement of marrow elements and resorption of bone to allow local tumor progression. Bone resorption and intraosseous tumor growth lead to bone pain, possibly due to necrosis, inflammation, and elevation of intraosseous pressure. In addition, loss of mechanical strength due to structural damage leads to pathologic fractures, unfortunately a common problem with metastatic carcinomas. Extensive bone resorption, or osteolysis, by metastatic tumors can lead to systemic hypercalcemia, an additional cause of morbidity and mortality.
Metastatic bone deposits tend to initially displace marrow elements preferentially, seeming to take the path of least resistance. For this reason, radiographs frequently appear normal, even with extensive metastatic involvement of bone. Radiographic evidence of bone lysis can require up to 50% loss of the mass of the trabecular bone to become readily apparent. Radionuclide bone scans, which detect subtle bony reaction to the advancing lesion, are a much more sensitive method of detection in most tumors. This is in part due to the local coupling of bone resorption and formation that occurs between osteoblasts (bone-forming cells) and osteoclasts (bone-resorbing cells).
Whenever osteoclasts resorb bone, they release so-called coupling factors locally from the bone matrix that putatively stimulate osteoblast progenitors to differentiate into osteoblasts in that area and form new bone (see Figure 1). Thus, even when tumors cause osteolysis and a net loss of bone locally, there is generally some reactive bone formation occurring also, accounting for radionuclide uptake on nuclear bone scans. The spectrum of cytokines and growth factors secreted by tumor cells within bone is also a determinant of the type of local bony reaction to the lesion. Metastatic carcinomas can have a predominantly lytic radiographic appearance (commonly seen with lung, renal, and thyroid carcinomas), a purely blastic appearance (prostate carcinoma), or a mixed pattern of bone lysis and blastic areas (breast carcinoma). We still have limited understanding of the factors that account for these differences, but several molecules have been identified that may be involved in determining the type of bony reaction to the lesion. Blastic prostatic tumors contain acid phosphatase, which is a glycosylated enzyme containing mannose-6-phosphate. The acid phosphatase may activate the mannose-6-phosphate/insulin-like growth factor (IGF-II) receptor of osteoblasts, which causes an anabolic effect and stimulates local bone formation (Ishibe, Rosier, & Puzas, 1991a, 1991b). In addition, calcitonin gene-related peptide (CGRP) in prostatic tumors may have osteoclastic inhibitory properties similar to calcitonin. Fibroblast growth factor (FGF) and bone morphogenetic proteins (BMPs) in prostate carcinoma could contribute to the abnormal bone formation observed (Harris et al., 1994; Nakamoto, Chang, Li, & Chodak, 1992). Although patients with blastic lesions may have serious problems with bone pain, they tend not to have fractures due to the sclerotic nature of the reactive bone within and around the metastatic lesions. Two theories have attempted to account for the mechanism of progression of lytic lesions in bone: direct osteolysis of bone by tumor cells, and stimulation of host osteoclastic bone resorption by the tumor cells. Recent evidence suggests that both mechanisms may be at work. Histologic study in our laboratory of tissue from lytic bony metastases, as well as primary round cell tumors such as Ewing's sarcoma and lymphoma with bone destruction, has consistently demonstrated the presence of large numbers of osteoclasts resorbing the bone. This phenomenon is not observed in predominantly blastic tumors. In addition, numerous researchers have identified three cytokines as important mediators of osteoclastic bone resorption: interleukin-1 (IL-1), interleukin-6 (IL-6), and TNF-*. Pathologic bone resorption is associated with local expression of these bone resorptive cytokines in a variety of pathologic conditions, including rheumatoid arthritis, osteomyelitis, wear debris-induced periprosthetic osteolysis, bone cysts, and multiple myeloma. TNF-* is particularly important in malignancy, because its secretion may be responsible for local normal cellular cytotoxicity, and it contributes to many of the severe systemic effects of disseminated malignancy, including constitutional symptoms like fevers, malaise, weight loss, and cachexia. In addition, TNF-* is secreted in a membrane-bound form that requires cleavage by an extracellular MMP in order to be released and activated (Gearing et al., 1994). Thus, antimetastatic therapy targeting MMPs may also treat systemic symptoms and metastatic bone resorption. In lymphoma of bone, we have demonstrated the presence of these cytokines immunohistochemically in the tumor cells in areas of bone lysis and osteoclastic stimulation (Hicks et al., 1995). Similarly, in lytic metastatic carcinomas and Ewing's sarcoma, we have observed expression of one or more of these 3 cytokines in all cases, possibly representing a significant component of the mechanism of bone destruction.
Figure 1. Coupling of Bone Resorption and Formation Through Bone Matrix Growth Factors.
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Direct tumor-mediated osteolysis remains a possibility, but efficient bone resorption is a fairly complex process requiring cell attachment, acidification of the space between the cell and the bone by means of a proton pump to dissolve mineral, and then secretion of acid-stable lysosomal enzymes to dissolve matrix. Thus, it is currently unclear to what extent tumor cells can duplicate the complex activities of the highly specialized osteoclast, although they can lyse bone readily by simply locally stimulating these host cells.
Tumors predisposed to metastasize to bone may do so via expression of cell surface receptors such as integrins or CD44, which may target bone matrix components for cell attachment. Integrins are heterodermic cell surface receptors composed of * and ß subunits that transduce signals through cytoskeletally associated kinases, and are expressed at high levels in most cells of mesenchymal origin. Cells of epithelial origin such as carcinomas normally express relatively low levels of these receptors, although upregulation may occur in malignancy. Integrins bind to matrix components containing a common arginine-glycine-aspartate (RGD) sequence. Bone contains numerous matrix proteins with RGD sequences, including collagens, fibronectin, and osteopontin. Synthetic or natural peptides containing the integrin binding motif (RGD), can block tumor attachment to bone in vitro and in animal models.
These molecules can also block osteoclastic bone resorption, since osteoclasts must attach to bone surfaces by an integrin known as the vitronectin receptor. CD44 is a hyaluronan receptor expressed in malignant cells that has also been suggested to have a role in cell attachment to bone matrix (Miyasaka, 1995). Thus, blockade of tumor attachment factors provides another potential route to therapeutic suppression of the occurrence or progression of bony metastases.
Parathyroid-hormone-related peptide (PTHrP), secreted by a large number of carcinomas, can cause hypercalcemia of malignancy due to stimulation of osteoclastic bone resorption. PTHrP probably acts through the same receptor as parathyroid hormone (PTH), stimulating osteoblasts to release IL-6, which then secondarily induces osteoclasts to resorb bone. This mechanism presumably drives the response of bone to PTH in regulating calcium homeostasis under normal physiological conditions, but is pathologically stimulated in cases of tumor secretion of PTHrP. In addition, evidence suggests that PTHrP expression may determine the propensity of a tumor to metastasize to bone. Kitazawa and Maeda (1995) recently demonstrated that, in a series of patients with metastatic breast carcinoma, the only patients who did not have bony metastases were those whose tumors did not express PTHrP. PTHrP expression, by inducing local osteoclastic bone resorption, may expose integrin ligands or other adhesion molecules in the bone matrix, facilitating malignant tumor cell attachment in this bone.
Experimental Therapeutics
Will this new information on mechanisms of metastasis help prevent or retard metastatic disease in patients? Preliminary data from a number of laboratory groups suggest that some relatively nontoxic agents that interfere with various aspects of metastasis may be efficacious adjunctive treatments. We have recently studied a malignant murine lung carcinoma cell line called line 1, which is readily transplantable in syngeneic mice due to low expression of HLA-DR antigens.
When injected intramuscularly in mice, these highly malignant cells rapidly develop into a primary tumor and predictably spread selectively to the surface of the lungs within a few weeks. The surface metastases can be readily quantified. Initial in vitro studies demonstrated production of MMP9 and a stromelysin by the line 1 cells by zymography. We chose two agents with theoretical MMP inhibitory activities after screening studies, minocycline and 9-cis-retinoic acid. Both of these agents potently suppressed (>90%) the MMP activity of the line 1 cells at pharmacologically relevant clinical dose levels. Next, we studied the cells using an in vitro metastasis model of invasion assay, that is, rate of tumor migration through a synthetic basement membranelike material called matrigel. Again, both retinoic acid and minocycline suppressed in vitro invasion by these cells by >90%, although the combination showed no additive effect. Subsequently, we inoculated mice intramuscularly with the line 1 cells in control (saline), minocycline, retinoic acid, or minocycline + retinoic acid groups treated daily by intraperitoneal injection. At sacrifice, while none of the treatments affected primary tumor size or necrosis, the treatment groups demonstrated a 93% decrease in lung tumor burden in the retinoic acid group, 74% decrease in the minocycline-treated group, and 57% decrease in the combination group, as compared with control (Pearson et al, 1996).
Further experiments clarified the lack of synergistic or additive effects of the MMP inhibitors, revealing that the effect of retinoic acid was mediated by a 7-fold stimulation of TIMP expression, abrogated by minocycline through a mechanism not yet defined. Thus, in vitro experimental observations in this particular model correlated quite well with in vivo effects. Both agents are relatively nontoxic, and could serve as adjunctive therapies to retard metastatic disease clinically. Continued research may uncover other combinations of MMP inhibitors, or agents that inhibit other phases of the metastatic process. These could be combined with MMP blockade to achieve additive or synergistic effects in the suppression of metastasis. Hydroxamates are under intensive development as other possible nontoxic MMP inhibitors for use in antimetastatic therapy.
Finally, another therapeutic target of enormous potential clinical benefit is the osteoclastic bone resorption of lytic bony metastases. Besides the theoretical use of anti-bone resorptive cytokine therapy mentioned earlier, other pharmacological agents exist that can prevent bone resorption in patients. Some of the earlier bone antiresorptive agents, such as etidronate and calcitonin, have not demonstrated clear clinical efficacy in malignancy except in cases of treatment of hypercalcemia.
However, the agents have not met the goals of reduction of bone pain and prevention of pathologic fractures. Bisphosphonates presumably act by adsorbing to the hydroxyapatite surfaces of bone and interfering with osteoclast function by an undefined mechanism. The first bisphosphonate, etidronate, also causes significant inhibition of mineralization. However, the recent development of a new generation of far more potent bisphosphonate antiresorptive agents with minimal or no inhibitory effects on bone formation offers exciting opportunities to ameliorate the morbidity of bone metastases. Following clinical trials of pamidronate, clodronate, and ibandronate, these agents have become well established for their efficacy in treatment of hypercalcemia in patients with metastatic cancer. However, several clinical trials have also reported average decreases in rates of pathologic fractures of 50%, significant decreases in bone pain, and even increases in mean survival with intermittent IV pamidronate therapy (Conte et al., 1995; Houston & Rubens, 1995). Current availability of orally active bisphosphonates such as alendronate could make therapy even more practical for cancer patients. In addition, some recent in vitro work suggests that biphosphonates may protect against tumor cell attachment to bone, and this hints at possible uses in the prevention of metastases.
Our group has recently begun treatment of patients with extensive lytic bone disease with oral alendronate therapy, with dramatic radiographic changes of lesions from lytic to blastic in a number of patients, and a decrease in bone pain and cessation of recurrent pathologic fractures in some patients. The rapid change from a lytic to a blastic radiographic appearance is not surprising given the recent advances toward elucidation of bone formation and resorption coupling mechanisms. In the context of an aggressive lytic malignancy in bone, enormous amounts of bone matrix are being dissolved by osteoclastic activity, releasing a large amount of anabolic growth factors into the local area. The catabolic, cytokine-mediated osteolytic effect of the tumor obviously dominates; however, upon sudden cessation of the profuse resorptive activity by initiating bisphosphonate therapy, the released growth factors which normally couple formation to resorption can cause rapid bone formation in and around the lesion. Although we need larger sample sizes and controlled clinical trials to confirm the efficacy of this treatment, the preliminary evidence suggests a distinctly beneficial effect for this otherwise relatively harmless therapy.
Conclusions
Recent insights into the mechanisms of metastasis, factors facilitating bony localization, and mechanisms of tumor-mediated bone resorption have revealed a number of novel and exciting potential approaches to the prevention or suppression of metastatic cancer. Clearly, the failure of current treatment methods to control metastasis and bone involvement underscores the need for a paradigm shift in our treatment of cancer. The use of nontoxic adjuvant antimetastatic therapies is a nascent field, but it shows tremendous promise. Currently, the most practical approaches for clinical application include the bisphosphonates and MMP inhibitors such as tetracyclines and hydroxamates. Early clinical results already suggest efficacy for hydroxamate MMP inhibitors and bisphosphonates in suppressing metastasis and bony progression. The possibility exists for even greater efficacy through combination with other more traditional methods of systemic treatment. Other work in angiogenesis inhibition, cell adhesion prevention, and immunotherapy suggests further options for developing novel programs of therapy that may ultimately bring about needed improvements in cancer care and prognosis.
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R.N. Rosier, R.J. O'Keefe, and J.E. Puzas are attending physicians in the Department of Orthopaedics at the University of Rochester/Strong Memorial Hospital in Rochester, NY. D.G. Hicks and L.A. Teot are attending physicians in the Department of Orthopaedics and the Department of Pathology at the University of Rochester/Strong Memorial Hospital in Rochester, NY.