Malignant bone resorption: Cellular and biochemical mechanisms

Annals of Oncology 3: 257-267, 1992. O 1992 Kluwer Academic Publishers. Printed in the Netherlands.

Review Malignant bone resorption: Cellular and biochemical mechanisms D. J. Dodwell Cookridge Hospital, Leeds, UK

Key words: bone resorption, bone metastases, osteoclast

Introduction

Post mortem studies reveal that 85% of women dying from breast cancer have bone metastases and corresponding figures for patients with prostate and lung cancer are 85% and 60% [94]. The high prevalence of these tumours means that clinicians treating patients with bone metastases face a large-scale clinical problem. The morbidity from skeletal involvement is considerable. Bone metastases are the commonest cause of cancer pain and in addition cause hypercalcaemia, immobility, pathological fracture, bone marrow failure and the neurological sequelae of nerve root or spinal cord compression. Currently, available treatments including external beam radiotherapy, systemic radionuclides, endocrine therapy and cytotoxic drugs may be temporarily effective but at some point skeletal destruction with its attendant morbidity continues. The osteoclast is responsible for the resorption of normal bone during remodelling and it is widely believed that tumour cells may cause bone resorption by stimulating osteoclasts. Advances in the understanding of bone and tumour biology have prompted the search for agents such as bisphosphonates, calcitonin or gallium nitrate which are not directly cytotoxic but by their 'anti-osteoclast' action may interfere directly with tumour-bone interactions. The development of these agents depends critically on a detailed understanding of the various ways that tumours may affect bone. The purpose of this review is to summarise the cellular and biochemical interactions between bone and tumour cells which result in the destruction of bone.

an integral functioning organ. This process is termed remodelling. In the light of the invariable histological observation that bone resorption always preceded bone formation, Frost proposed the remodelling concept of coupled bone resorption and formation [30]. The process he postulated occurs as follows: after a certain amount of bone is removed by osteoclastic resorption and the osteoclasts have moved away from the resorption site, a reversal phase takes place. A cement line is laid down by osteoblasts which go on to synthesize, and then mineralize, bone matrix. Clearly close control of this crucial process is essential to maintain skeletal integrity and an understanding of the biochemical basis of this coupling has been the goal of bone biologists for many years. This is of particular importance clinically, because many disease states affecting bone including malignant bone destruction may result from a relative uncoupling of this process with an unregulated increase of resorption and/or formation [63].

Mechanisms of malignant bone resorption

Bone destruction in osteolytic metastases may occur by two methods. Bone may be destroyed directly by tumour cells, or tumour cells may stimulate host cells, principally osteoclasts, to resorb bone. The two methods that have been widely used to distinguish the relative contributions of osteoclasts and tumour cells to bone destruction are histopathological studies and cell and tissue culture experiments.

The compartmental mechanism of bone turnover

Histopathology

Bone formation and resorption proceed throughout life. Clearly bone formation is more rapid during the phase of skeletal growth in embryonic or early life at which time the term modelling is used. During adult life control of the processes of bone resorption and bone formation remain essential to maintain the skeleton as

Milch and Changus studied histological sections from 241 bone specimens, obtained at post mortem, from patients known to have skeletal metastases [59]. They found little evidence that osteoclasts were directly osteolytic. They did however report the presence of multinucleated giant cells, which they termed 'tumour

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giant cells', in many of the specimens. These appeared to be present more commonly in areas where bone formation was more conspicuous than bone destruction. However, at the time this study was performed specific histochemical and immunocytochemical stains for osteoclasts were not available and osteoclasts were not definitively established as a distinct cellular entity. Galasko reported a series of bone biopsies obtained at autopsy from the vertebrae of 68 patients who had died from various malignant diseases [34]. Examination of this material suggested that there were two distinct mechanisms for bone destruction. In many instances the bone surface was lined by numerous osteoclasts with local resorption occurring in the lacunae around each osteoclast. In other sections the bone edge had a trabeculated appearance suggesting that these were osteoclastic lacunae, although no osteoclasts were seen. This occurred with all types of tumour. In other sections, however, where there was gross bone destruction there were only residual spicules of bone surrounded by tumour cells but not osteoclasts. These observations in human biopsy material matched those in a series of rabbit experiments using the VX-2 carcinoma. A suspension of VX-2 cells was injected into the tibia or onto the periosteal surface of New Zealand white rabbits. The first change seen in these animals, at twentyfour hours, was osteoclastic proliferation separated from the tumour by a small amount of fibrous stroma. As the tumour grew, osteoclasts proliferated. When the tumour was injected onto the periosteal surface, osteoclastic destruction of the cortex, extending from the periosteum to the endosteal surface occurred, osteoclasts always preceding the tumour. However, once the tumour had grown sufficiently large to envelop a residual trabeculum of bone, osteoclasts disappeared but bone destruction continued. These observations in experimental models and in human biopsy material suggested that destruction of bone, associated with metastatic invasion, involved at least two mechanisms, the initial and apparently the more quantitatively important was mediated by osteoclasts. The second, which seemed to operate in the later stages of bone destruction was mediated directly by tumour cells. However, in contrast to these observations, Hulth and Olerud, using the same experimental model, reported (on the basis of histopathological observations) that the majority of bone destruction seemed to be caused by the VX-2 tumour cells themselves, and osteoclasts played a less significant role in osteolysis [44]. Support for the two stage process of bone destruction was provided by a further histological study by Carter, who studied the morphological aspects of direct bone invasion in one hundred patients undergoing resection of the mandible or wide-field laryngectomy [26]. From this study he drew the following conclusions: i) Bone destruction appeared to be a two stage process as described by Galasko [34]; ii) osteoclasts appeared to be incapable of destroying contiguous areas of cartilage; iii) cartilage destruction was mediated by tumour

cells acting alone, or in company with mixed inflammatory cells - osteoclasts were not involved in this process; iv) if metaplastic ossification occurred (usually in the larynx) such regions were destroyed in the same way as skeletal bone with local osteoclasts playing a prominent role; v) there was a consistent spatial proximity between tumour cells, osteoclasts and the bone surface. It should be noted that this study was performed on bone invaded directly by contiguous extension, predominantly from squamous tumours of the head and neck, and may not accurately reflect the bone destruction seen in bone metastases in distant sites, which have seeded haematogenously. The inability of osteoclasts to resorb poorly mineralised bone, cartilage or osteoid has been independently confirmed [50]. A study of the reactive changes occurring near vertebral metastases from various histological types of bronchogenic carcinoma was reported to show apparent metabolic activation of bone lining cells with exuberant osteoclastic and osteoblastic activity in close proximity to the invading neoplasms [22]. Cell and tissue culture

Cell and tissue culture systems have been used to examine the osteolytic capabilities of tumour cells and osteoclasts. There is a great deal of evidence that products which are released from tumours may stimulate osteoclastic activity. However, there is relatively little published work on the effects of cultured tumour cells, acting alone, on bone resorption in vitro. Eilon and Mundy reported that release of 45Ca and 3 H-proline from prelabelled live and dead fetal rat long bones occurred when these were cultured with MCF-7 (breast cancer) cells or the supernatant media from these cells [25]. Histological examination of the resorbing live bones which had been cultured with MCF-7 media showed no increase in the number of osteoclasts adjacent to the endosteal bone surfaces, whereas bones cultured with prostaglandin E-2, as a positive control, showed a greatly increased number of osteoclasts. To confirm that osteoclasts were not required for these cultured breast cancer cells to resorb bone, live bones were cultured with phosphate and cortisol (inhibitors of osteoclast function) and then treated with the medium from MCF-7 cells. No inhibition of the calcium release was seen, in contrast to the effects of these inhibitors on live bones stimulated with prostaglandin E-2. Similarly the authors reported that prostaglandins were not the cause of the calcium and hydroxyproline release because it was not inhibited by indomethacin and additionally the medium from MCF-7 cells did not contain immunoreactive prostaglandins of the E series. The authors also examined the effects of a number of different breast cancer cell lines and observed that they all caused bone resorption independently of osteoclasts, whereas normal lymphocytes, lymphocytes from

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a patient with chronic lymphatic leukaemia, and cell lines developed from ovarian cancer, lung cancer and a chondroblastoma showed no osteolytic activity in this system. Although this study provides some evidence that rumour cells may be directly osteolytic it is difficult to comprehend the mechanisms by which tumour cells destroy bone independently of osteoclasts. Optimal dissolution of bone mineral requires an acid pH and although the capability to produce a proton pump may reside within the genomic DNA of tumour cells, as they are derived ultimately from the same cell as osteoclasts, there are no published data demonstrating that malignant cells have this ability. Study of the relative contributions of osteoclasts and tumour cells to bone destruction is of critical importance to the place of bisphosphonate therapy and other treatments designed to suppress excessive osteoclastic resorption in the systemic therapy of skeletal metastases. The study of malignant bone resorption in osteopetrotic animals would facilitate the further examination of the role of the osteoclast in this process.

found on the osteoclast cell membrane but are found on osteoblasts. Thus in addition to its role in bone formation the osteoblast may be of critical importance in the hierarchical control of bone resorption. One of the mechanisms whereby osteoblasts could control bone resorption is by the initial secretion of type I collagenase to facilitate the removal of the nonmineralised layer on the surface of bone matrix, thereby allowing osteoclast access to the underlying mineralised bone (Fig. 1). There is now evidence that osteoblastic cells produce and secrete procollagenase. This has been demonstrated in murine woven bone and by osteoblastic cells or cell lines isolated from mouse or rat bone [41,67, 83, 84]. Osteoblast-like cells have been shown to produce procollagenase in response to bone resorbing agents including PTH [84], retinoic acid [68], 1, 25 (OH)2 vitamin D 3 [41], EGF [68], and monocyte conditioned medium [41]. Recent observations from Pfeilschifter et al. have demonstrated that mature osteoblasts and osteoblast precursors derived from fetal rat calvariae showed an increase in plasminogen activator activity after incubation with PTH or prostaglandin E-2 and a decrease in Biochemical mechanisms activity following incubation with transforming growth factor beta (TGF-beta) [71]. These effects were accomProteolysis and bone resorption panied by a corresponding inverse effect on plasminoType I collagen comprises 90% of the protein content gen activator inhibitor (PAJ) activity. The most active of adult bone and accounts for 20% of the weight of cells, in terms of PA activity, appeared to be the prefresh bone. Collagenolysis is therefore an integral com- osteoblasts rather than the mature osteoblasts. These results were supported by Catherwood et al., who ponent of bone resorption. The osteoclast, which is the major bone resorbing demonstrated a 13-fold increase in mRNA levels for cell, does not apparently produce type I collagenase tPA in rat calvarial osteoblasts stimulated by PTH [17]. Because the plasminogen activator system is an imbut does secrete lysosomal acid proteinases including collagenolytic cysteine proteinases, namely the lysoso- portant factor in the control of the proteinase cascade system, changes in plasminogen activator activity may mal cathepsins. However, cysteine proteinases such as the cathepsins cause magnified effects on the activity of type I colcan only cleave type I collagen in the telopeptide region lagenase which is secreted in an inactive form but is (the segment of the molecule flanking the triple helical activated by plasmin, generated by increased PA levels. There is now much evidence for the involvement of structures) and, unlike type I collagenase, are unable to cleave type I collagen within the triple helix itself. Thus, type 1 collagenase in bone resorption and support for collagenolysis is accomplished at a relatively low effi- the hypothesis put forward by Chambers (19] who suggested that the unmineralised osteoid layer covering ciency by cysteine proteinases acting alone. Observational studies of osteoclasts also cast doubt bone surfaces 'protects' bone from osteoclastic resorpupon the role of the cathepsins as the major cellular tion. This osteoid surface must be removed by proenzymes responsible for the degradation of bone type I teinases prior to osteoclast attachment and activation collagen. Despite the powerful resorbing effects of (Fig. 1). osteoclasts on mineralized bone matrix, their action on However, this experimental evidence is derived from the unmineralised matrix covering bone surfaces is systems comprising of fetal or neonatal woven bone a infrequent in vivo [56] and is inefficient in vitro [18]. temporary mineralised tissue which, in normal growth Other evidence for the involvement of type I col- and development, is destroyed and replaced by mature lagenase in bone resorption stems from the increasing lamellar bone. In general the role of collagenase in the awareness of the important role of the osteoblast or physiological and pathological resorption of mature osteoblast-like cell in the control of bone resorption adult bone is much less well investigated. (Fig. 1). Co-culture experiments have demonstrated In summary, although there is much data to suggest that the majority of bone resorbing agents appear not that collagenase may be of major importance in bone to affect osteoclasts directly, but by the mediation of resorption the precise roles of proteinases, particularly osteoblast-like cells [19]. Receptors for bone resorbing type I collagenase in the osteolysis of malignancy agents, including parathyroid hormone (PTH), are not remain undetermined.

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ture has evolved concerning the role of cytokines in both physiological and pathological states. The revolution in understanding of information transThe interactions between malignancy and bone fer between cells at a paracrine or autocrine level has which result in the clinical manifestations of bone come about since the discovery and isolation of a metastases are reported to occur predominantly by growing number of polypeptide growth factors and paracrine processes, mediated by a number of these cytokines. In addition to these, the control of various cytokines. In addition skeletal pathology (and particuorgans, at the systemic level, is achieved by alterations larly tumour-induced hypercalcaemia) may result from in their endocrine environment. These two systems of the distant release of tumour products and this process cell and tissue control act in concert to maintain nor- may thus be regarded as a disturbance of endocrine mal homeostasis and growth during fetal, infant and skeletal homeostasis. Different tumour types affect boadult life. Bone is no exception to this general rule and ne in different ways and a discussion of the mechanisms there is increasing evidence that the skeleton is heavily of osteolysis in relation to common tumour types with influenced by endocrine and paracrine factors. Bone emphasis on the role of cytokines, including the polybiologists have studied the effects of many hormones, peptide growth factors, prostaglandins, 1,25-dihydrocytokines and polypeptide growth factors in the various xyvitamin D 3 and parathyroid hormone related peptide model systems of bone and a vast and complex litera- (PTHrP) will be discussed here. Although these mechaParacrine processes in osteolysis and hypercalcaemia

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nisms may be broadly categorised for the sake of clarity is must be remembered that a combination of processes may exist within a particular clinical situation (Fig. 1).

1,25 vitamin D was seen [10]. These data suggest that the bone resorbing activity of IL-1 and TNF, but not PTH or 1,25 vitamin D, are mediated by the production of IL-6. IL-6 has also been shown to be an autocrine/paracrine growth factor for Pagetic osteoMultiple myeloma clasts [81]. Bone is invariably affected in patients with this disease Although there is little doubt that excessive bone and although multiple myeloma is classically character- resorption contributes to the osteolysis in myeloma, ised by lytic bone lesions without accompanying scle- additional mechanisms are required to explain the rosis the disease commonly produces a diffuse 'osteo- skeletal pathology that is seen to occur. porosis' rather than discrete skeletal lesions. Such In physiological circumstances, given the normal patients have been reported to have a poorer survival coupling between bone resorption and bone formation, compared to those with extensive lytic disease [93]. it would be expected that this excessive bone resorpHistological studies have confirmed that osteoclastic tion would be accompanied by an increase in osteobone resorption is increased in proximity to myeloma blastic bone formation. In a survey of 118 bone biopcells [99]. Mundy et al. reported that myeloma cells sies taken from patients with myeloma, Valentin-Opran produced a form of osteoclast activating factor (OAP) et al. found that the number of osteoid surfaces and the [64]. Durie et al. [24] found that the in vitro production percentage of trabeculae that exhibited tetracycline of this substance significantly correlated with extent of labelling (suggesting increased bone formation surbone destruction in a group of 33 patients with multi- faces) were increased compared to control patients [99]. However lower calcification rates and reduced ple myeloma. Garrett et al. isolated tumour cell lines from a pa- thickness of osteoid seams were also found, and overall tient with multiple myeloma and osteolytic lesions and the appearances suggested a reduced activity for each found that most of the bone resorbing activity was sup- osteoblast. pressed by neutralising antibodies to lymphotoxin Bataille et al. reported a similar study in 21 patients (tumour necrosis factor beta) [37]. Similar results were with myeloma (MM) [3]. They identified by histomorseen from four established cell lines from a further phometric criteria, two distinct mechanisms of bone three patients. They concluded therefore that lympho- destruction, one occurred in patients with lytic bone toxin production was related to osteoclastic bone re- metastases and was characterised by increased bone sorption and hypercalcaemia in myeloma. resorption with normal to low bone formation (unbalIs is now accepted that lymphotoxin is one compo- anced MM) whereas patients without lytic lesions had nent of 'OAF although tumour necrosis factor alpha increased bone resorption with increased parameters and interleukin-1 may have contributed to the bone of bone formation (balanced MM). This histomorphoresorbing activity ascribed to 'OAF in the past [9, 43] metric distinction was confirmed by measurement of serum levels of the osteoblast product osteocalcin. and the term 'OAF' should no longer be used. It has recently become evident that interleukin-6 Levels were significantly higher in patients with balmay also be an important factor in the causation of the anced MM than in those with unbalanced MM. Interbone resorption induced by myeloma. IL-6 refers to a estingly the findings of Smith et al. (93] that myeloma gene product that was characterised initially as beta-2 patients with diffuse 'osteoporosis' have a poorer surinterferon, a 26-kilodalton protein produced by human vival compared to those with lytic disease were confibroblasts, shown to be identical to B-cell stimulatory firmed by this study. factor-2 and hybridoma/plasmacytoma growth factor This inhibition of osteoblast activity seen in those [87]. It is expressed at high levels in the organs of nor- patients with lytic disease is compatible with the mal individuals [98] and has been shown to support pathophysiological properties of lymphotoxin which haemopoeitic stem cells in vitro in conjunction with has been shown to inhibit bone formation in culture [9]. IL-3 [66] and IL-4 [77]. However myeloma cell cultures have also been shown This cytokine is also an autocrine growth factor to release an 'osteoblast inhibitory factor' which shares for myeloma as myeloma cells in culture have been similarities to TNF-beta, although the precise nature of demonstrated to produce excessive amounts of IL-6 this cytokine has yet to be characterised [26-28] and [10] and respond to this factor by proliferation [90]. the definitive mechanisms by which myeloma causes IL-6 has been shown to cause hypercalcaemia in vivo osteolysis remain to be determined. and enhances the potency of IL-1 and TNF by two orders of magnitude in vitro [10]. Endogenous IL-6 Focal osteolytic disease production increased when mouse calvarial cultures were treated with IL-1 or TNF and neutralising anti- Although multiple myeloma may be considered a form bodies to IL—6 (which did not cross react with IL-1 or of focal osteolytic disease, this term is usually applied TNF) completely abolished the bone resorbing activity to bone metastases from common solid cancers. These of exogenously administered IL-1 or TNF. In contrast are most commonly from breast, prostate and lung no inhibition of bone resorption induced by PTH or primaries.

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Histological studies have demonstrated that both osteoblastic and osteoclastic activity are increased in the proximity of tumour cells in patients with lung cancer [22] and breast cancer [34]. Although osteoblastic rather than osteolytic metastases are associated with skeletal prostate cancer there is nevertheless histological and biochemical evidence that osteoclastic bone resorption is increased in these patients [69]. It is however not known with certainty which cytokines are responsible for the focal stimulation of osteoclastic activity seen in these conditions, particularly as these common tumours, or cell lines derived from these tumours, have been shown to produce a number of substances including cytokines, growth factors and prostaglandins, most of which have been demonstrated to stimulate bone resorption in fetal or neonatal bone fragment culture systems and cause hypercalcaemia in experimental animals. Prostaglandins, particularly those of the E series, were believed to be implicated in this process. Many tumours produce more prostaglandins than the normal tissues from which they arise [4]. Similarly it has been known for many years that prostaglandins are potent stimulators of bone resorption in organ cultures [52] but interestingly do not stimulate isolated osteoclasts

also correlated with early death [4, 5]. It was suggested at that time that tumours with high levels of prostaglandin production had a predilection to metastasize to bone. However other workers [33, 100] found no correlation between tumour prostaglandin production and site of distant recurrence or survival time. Indeed, after further follow up of their original study Bennett et al. found that, although there was a correlation with prostaglandin-like material production and the presence of bone metastases near the time of surgery, levels of prostaglandin production within tumours did not predict later recurrence in bone [6]. Similarly there was no relationship between tumour prostaglandin-like material and the grade of malignancy, tumour type, amount of fibrous tissue invasion, invasion of blood vessels and lymphatics or presence of plasma cells. Disease free survival appeared longest in patients with an intermediate level of production of tumour total prostaglandin-like material. Clearly one of the problems with these types of studies is that assays performed on tumour extracts cannot determine the levels of differential production of prostaglandins from tumour cells or associated host cells.

Seyberth et al. reported that inhibitors of prostaglandin synthesis were useful in the treatment of patients with hypercalcaemia from various non-haemato14 Support for the role of prostaglandins as mediators logical tumours [88]. However, the weight of clinical of tumour-induced bone resorption came from a series evidence would now refute this and suggests that indoof co-culture experiments performed by Galasko et al. methacin and similar drugs are not useful for the treatwho studied the effect of the prostaglandin synthetase ment of hypercalcaemia and do not decrease bone reinhibitor indomethacin on osteolysis [35]. There was a sorption clinically as assessed by urinary hydroxyreduction of osteolysis (as assessed by the release of proline excretion [21]. In summary, therefore, there is a large and often concalcium from neonatal calvarial cultures after co-incuflicting literature concerning the role of prostaglandins bation with a 1 mm cube of the VX-2 carcinoma). Simiin the causation of malignant bone resorption. No unilar results were seen using samples of human breast fying hypothesis exists to explain the role of prostacancer. When used in vivo aspirin and indomethacin glandins and prostaglandin metabolites in bone and tuhave been demonstrated to inhibit the growth of osteomour cell biology. In general, since the discovery and lytic tumour deposits and hypercalcaemia in rats with isolation of other cytokines and polypeptide growth the Walker tumour [73]. factors, which are felt to be more likely candidates for Because of the ability of tumours to produce large chemical mediators promoting tumour osteolysis interamounts of prostaglandins [4] many workers have atest in prostaglandins, in this respect, has tended to tempted to correlate tumour prostaglandin production wane. and clinical outcome. In view of the effects of prostaProcathepsin D is produced by a number of breast glandins on bone resorption others have investigated cancer cell lines and is unusual amongst bone active the 'osteolytic potential' of tumours (predominantly breast cancers) and prostaglandin synthesis. In this factors in that it is reported to activate osteoclasts regard, Powles et al. [74] found that of 38 human mam- directly [65] whereas virtually all other hormones mary carcinomas, 23 had significant in vitro osteolytic (other than calcitonin) and cytokines exert their actions activity (as assessed by calcium release from cultures of on osteoclasts via intermediary osteoblast-type cells. neonatal calvaria co-incubated with tumour samples) The clinical relevance of this protease precursor is unand all of their patients presenting with bone metas- known, but it is likely to have local rather than systemic tases or hypercalcaemia had 'osteolytically active' tu- effects on bone. mours. Over a three-year follow-up period bone metasTransforming growth factor alpha (TGF-alpha) has tases did not develop in any of their 15 patients with been particularly implicated in the paracrine stimula'osteolytically inactive' tumours. Bennett et al. reported tion of osteolysis. TGF-alpha is a polypeptide originally that the production of prostaglandin-like material by described as an inducer of uncontrolled growth in cerhuman primary breast carcinomas was higher in patients tain non-neoplastic cell lines [23]. TGF-alpha has been with scintigraphic evidence of skeletal metastases and described in normal human platelets and epithelial

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cells [53] and has forty percent sequence homology to epidermal growth factor (EGF) sharing the same cell surface receptor [58]. TGF-alpha is produced by cultured breast cancer cell lines and is present in high concentrations in patients with breast tumours [38]. In support of a role for TGF-alpha as a mediator of bone resorption in physiological and pathophysiological states, Ibbotson et al. [45, 47] showed that in a variant of the rat Leydig cell tumour model of the humoral hypercalcaemia of malignancy, tumour extracts and tumour conditioned media contained a macromolecular bone resorbing factor which co-eluted on column chromatography with TGF-alpha. In addition the bone resorption stimulated by tumour conditioned media was blocked by an anti-serum which blocked the binding of EGF to its receptor suggesting that the tumour-derived bone resorbing factor was dependent upon the availability of EGF/TGF-alpha receptors for its activity and therefore consistent with it being TGF-alpha. Tashjian et al. went some way in validating such a hypothesis by demonstrating a significant rise in plasma calcium concentration in mice given EGF and TGF-alpha [97]. Such a rise was invariably accompanied by an increase in systemic levels of a metabolite of prostaglandin E-2. Concurrent administration of indomethacin, whilst abolishing the increase of PGE-2 metabolite level showed no inhibition of the elevation in plasma calcium. This illustrates the difficulty in relating prostaglandin metabolism to the effects seen in bone cultures after administration of exogenous agents such as growth factors. TGF-alpha has been shown to stimulate bone resorption and inhibit formation in a dose dependent manner in a 45Ca labelled fetal long bone culture system and the neonatal mouse calvarial assay [46]. The activity was ten-fold greater than that of EGF tested in the same system although the time course was markedly different, in that the resorption caused by TGF-alpha occurred within forty-eight hours whereas the effect of EGF was slower in onset. Although the function of TGF-beta is believed to be largely anabolic with respect to bone [36, 70] it has been demonstrated to increase bone resorption under certain conditions [96]. TGF-beta is a polypepude growth factor consisting of two identical twelve kilodalton chains linked by disulphide bonds. It is produced by a variety of normal tissues and tumours (79, 80]. Although abundant in numerous tissues TGF-beta is present in particularly high concentrations in bone matrix. Linkhart et al. showed that conditioned medium from Sk-Leuci-6 cells (established from a large cell anaplastic lung tumour from a patient with hypercalcaemia) contained TGF-beta, which co-purified with its osteolytic activity [55]. Additionally no displacement of iodinated EGF binding to cell receptors, or stimulation of cyclic AMP formation in rat osteosarcoma cells occurred, suggesting that the conditioned medium did not contain TGF-alpha or EGF. Other cytokines have also been implicated in certain

tumour types. Cell lines obtained from tumours from patients with hypercalcaemia have been shown to contain interleukin-1, the most potent bone resorbing cytokine [29]. Sato et al. demonstrated the production of IL-1-alpha-like and colony stimulating factor activity by a squamous cell carcinoma of the thyroid derived from a patient with hypercalcaemia and leucocytosis [85]. IL-1 activity has also been identified in leukaemic cells from patients with adult T-cell leukaemia, a condition associated with lytic bone lesions and hypercalcaemia [91]. There are also a number of reports relating the production of bone resorbing factors by tumours from hypercalcaemic patients and animals in which there is also increased production of colony stimulating activity and consequent leucocytosis [54, 86]. The colony stimulating activity was found to co-elute with the bone resorbing activity, but the physiological role of the colony stimulating factors in osteoclast maturation is illunderstood despite the link between this process and the differentiation of cells from the haemopoietic lineage [57]. There are also, in all likelihood, a number of as yet uncharacterised factors which may be released from tumours to perturb cell biology. The predominantly sclerotic nature of skeletal prostate cancer has fuelled the search for osteoblast stimulating factors and indeed it has been shown that both hyperplastic and adenocarcinomatous prostate tissue release a substance which is mitogenic for osteoblasts [49]. More recently other factors which are mitogenic for osteoblasts have been identified in prostate carcinoma cells including bone morphogenetic protein (BMP) types 3 and 4 [39, 40], urokinase type plasminogen activator [75] and basic fibroblast growth factor (bFGF) [51]. Humoral osteolysis This term is used to indicate the stimulation of osteoclastic bone resorption which is induced by the distant release of tumour products and is therefore a form of endocrine paraneoplastic syndrome. This mechanism explains the syndrome of 'humoral hypercalcaemia' where malignant hypercalcaemia may occur in the absence of bone metastases (occurring commonly in hypernephroma and squamous cell lung cancer). There is much interest in this phenomenon since the relatively recent recognition of parathyroid hormone related protein (PTHrP) and it is of interest to study the evolution in understanding that has taken place following the recognition of this peptide. Albright raised the possibility that the hypercalcaemia of malignancy may be due to production of a parathyroid hormone (PTH)-like substance by the cancer [1]. Following this, the term 'ectopic PTH secretion' came into common usage to describe the syndrome of patients with cancer who had a high plasma calcium. After the development of the first radioimmunoassay for PTH, Berson and Yallow found significant eleva-

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tions of plasma PTH levels in an unselected group of patients with bronchogenic cancer [8]. Over the next few years several studies were published reporting elevated PTH levels in plasma or tumour extracts from patients with malignant hypercalcaemia [12, 89]. However, with the improvement in radioimmunoassay techniques, in the early 1970's, doubt began to emerge about the involvement of PTH in this syndrome. Two groups of workers published results indicating that the circulating immunoreactive PTH measured in cancer patients differed from authentic PTH [7, 78]. Powell and colleagues found that PTH could not be detected in the plasma or in tumour extracts in several patients with humoral hypercalcaemia despite the ability of tumour extracts derived from these patients to stimulate bone resorption in vitro [72]. The most conclusive evidence which demonstrated that PTH itself was not usually involved in the pathogenesis of malignant hypercalcaemia came from the use of complementary DNA probes. Using this technique it was demonstrated that no messenger RNA for PTH was being manufactured by many of the tumours considered as prime candidates for ectopic PTH secretion [92]. PTHrP was isolated, purified and cloned from mammalian cells [61, 95] and these original studies have allowed the detection and measurement of this protein in human blood and other tissues. Since that time cell culture and immunohistochemical studies have demonstrated that many animal and human tumours and cell lines produce PTHrP. The pathophysiology of hypercalcaemia in breast cancer has also recently been re-evaluated and it is now evident that, despite the high prevalence of bone metastases in this disease, 'humoral' mechanisms are more commonly involved than previously realised [20, 42, 48]. PTHrP has also been identified by immunohistochemical studies in tumour tissue in 60% of a series of 99 normocalcaemic breast cancer patients [60]. Further workers have attempted to identify the possible role of PTHrP in normal bone cell physiology. Fukayama et al. studied the comparative effects of PTH and PTHrP on bone turnover by assessing resorption in the neonatal calvarial model and by studying cyclic AMP production in an osteosarcoma cell line [31]. They found that there was a dose dependent production of cyclic AMP in these osteoblast surrogate cells after stimulation with PTH or PTHrP but that a second challenge with either peptide caused a blunted response suggesting receptor down-grading or decoupling of the receptor from adenylate-cyclase. PTHrP was three times more potent than PTH in its stimulation of bone resorption and this effect was apparently independent of prostaglandin production, as it could not be prevented by indomethacin. Similar results were reported by Raisz et al. [76]. Canalis et al. assessed the effects of PTHrP on bone formation in cultures of fetal rat calvariae [14, 15). Continuous treatment with this peptide stimulated

DNA synthesis but inhibited 3H-proline incorporation into collagen by approximately fifty percent. It contrast, transient exposure at low concentrations caused a twofold increase of 3H-proline incorporation into both collagenous and non-collagenous proteins. The median level of insulin-like growth factor 1 (IGF-1) was increased simultaneously by a factor of 3.5 and neutralising antibodies to IGF-1 prevented the stimulatory effects of transient exposure to PTHrP on bone collagen synthesis. The authors concluded that continuous treatment with PTHrP inhibited - whereas transient treatment stimulated - collagen synthesis and that the stimulatory effect appeared to be mediated by enhancement of local production of IGF-1. These results highlight the difficulty in ascribing a physiological role to a particular cytokine, since it has been widely demonstrated that biological effects may depend on other confounding variables such as time course, concentration and the effect of repeated administration. In addition to being produced by malignant tumours PTHrP is found in normal keratinocytes, lactating mammary tissue, placenta, parathyroid gland, central nervous system tissue and a number of other sites suggesting a widespread (but as yet unknown) physiological role for this polypeptide [13]. In summary although it is reasonably certain that PTHrP is a major circulating hypercalcaemic factor in patients with the humoral hypercalcaemia of cancer and may have other homeostatic actions, its role in the malignant resorption of bone in normocalcaemic patients is not defined. There is some evidence that tumour production of activated vitamin D3 may be responsible for hypercalcaemia occurring in certain types of lymphoma particularly the rare HTLV-1 associated adult T-cell leukaemia/lymphoma, a rapidly fatal condition characterised by lymphadenopathy, hepatosplenomegaly, lymphocytosis and hypercalcaemia. Elevated serum levels of this hormone are reported, in addition to these clinical features, and the absence of bone involvement despite increased osteoclastic activity on bone biopsies suggests a humoral mechanism [11, 62, 82]. However there is some dispute concerning the pathophysiological role of vitamin D even in this small patient group as Fukumoto et al. [32] have reported a group of hypercalcaemic patients with HTLV-1 leukaemia/lymphoma with low vitamin D levels and a high urinary excretion of cAMP suggesting that the hypercalcaemia may be caused by a PTH-like factor. Conclusions

Despite the advances in understanding of both normal and pathological bone turnover that have been discussed, the final common pathway — if such a thing exists - of malignant bone destruction remains unknown. One of the major reasons for this is the relative

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lack of good experimental model systems of mature adult bone. One method that has been used to determine the relative importance of the various cytokines that have been discussed, as possible mediators of bone resorption, is the powerful technique of in situ hybridisation. This technique has already provided evidence for the involvement of canine distemper virus in Paget's disease [Anderson, personal communication]. However, at present, the precise roles of cytokines and growth factors, the importance of proteolysis and the comparative resorptive capabilities of tumour cells and osteoclasts, in malignant bone resorption are unclear and the rational development of agents designed to interfere with malignant processes in bone is hampered by this lack of information. Significant improvements in the treatment of bone metastases will only come about by a greater understanding of bone and tumour biology.

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Correspondence to: Dr. D. J. Dodwell Cookridge Hospital Leeds, LS 16 6QB, U.K.