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Professor Øyvind S. Bruland
 

Professor Øyvind S. Bruland

 

Pathophysiology of bone metastases and mechanism of action of bisphosphonates


Introduction

Advanced cancers frequently spread to bone, and the resulting bone destruction is associated with a variety of skeletal complications, including pathologic fractures, bone pain, impaired mobility, spinal cord compression, and hypercalcemia. It is estimated that world-wide, more than 1.5 million patients with cancer have bone metastases. Current treatment options for patients with bone metastases include radiation therapy, surgery, bisphosphonates, and analgesics, in addition to standard anticancer therapy. The primary goals of therapy are to minimize bone pain and morbidity and improve mobility and quality of life.

Bone is not an inert organ. During adult life, normal bone undergoes a continuous remodelling process of resorption and formation. This is normally a tightly coordinated process in which initial osteoclast resorption takes place in discrete ‘packets’, known as bone remodelling units, over a period of about 8 days. This is followed by a more prolonged phase of bone formation (over about 3 months) mediated by osteoblasts to repair the defect.

Pathophysiology of bone metastases
In normal healthy bone there is a steady-state balance, or "coupling", of osteoblastic bone formation and osteoclastic bone resorption, which is lost when tumour cells enter the bone microenvironment. In vitro, breast cancer cells produce parathyroid hormone-related protein (PTHrP), (Guise, 2000) which stimulates osteoclastic resorption by increasing osteoblast and stromal cell production of receptor activator of nuclear factor ę B (RANK) ligand. RANK ligand binds to its receptor RANK on osteoclast lineage cells inducing differentiation into mature osteoclasts and stimulation of osteoclast activity.

Within normal bone, the secretion of osteoprotegerin (OPG) by osteoblasts neutralises RANK ligand, and prevents the stimulatory effects of RANK ligand on osteoclasts. However, in bone harbouring cancer cells there is loss of regulatory control. The imbalance is further compounded by release of transforming growth factor- beta (TGF-â) and insulin-like growth factor (IGF-1) by resorbing bone.

These have been found to act as survival factors for breast cancer cells, promoting tumour production of PTHrP, (Guise, 2000) and thus allowing a perpetuating a vicious cycle of osteolytic bone destruction (Figure 1).

Fig.1 Tumor cell and bone symbiosis

Prostate cancer tends to cause osteoblastic lesions in bone. It is hypothesised that osteoblastic lesions are formed by intense osteoblastic activity, preceded by osteoclastic bone resorption, as indicated by increased levels of urinary markers of osteolytic bone resorption in prostate cancer patients. (Garnero et al., 2000) Conversely it is also possible that prostate tumour cells can induce metastatic bone lesions that do not involve osteoclastic activity. A recent study (Lee et al., 2002) found that prostate cancer cells (PC-3) implanted into the tibia of SCID mice caused osteolytic lesions, possibly through secretion of RANK ligand. However, when another prostate cancer cell line (LAPC-9) was used, osteoblastic lesions developed even when no osteoclasts were present.

The growth factor endothelin-1 (ET-1) may also be involved in prostate cancer bone metastases. In comparison to patients with either cancer confined to the prostate or normal controls, circulating levels of endothelin-1 are increased in patients with osteoblastic bone metastases from androgen-refractory prostate cancer (Nelson et al., 1995). Endothelin-1 stimulates osteoblasts, and inhibits osteoclast activity in animal models, whilst antagonists of endothelin have been found to inhibit bone formation in vivo. Furthermore a recent study (Granchi et al., 2001) found that endothelin-1 production by prostate cancer cells is reduced by androgens but stimulated in androgen-insensitive prostate cancer cells by factors, (such as TGF-â). PTHrP is probably also involved in the pathogenesis of prostate cancer bone metastases, as co-expression of PTHrP and its receptor has been found in both the primary tumour and bone metastases of patients with prostate cancer (Bryden et al., 2002).

Bone marrow stromal cells are important in the pathogenesis of myeloma bone disease. Interleukin-6 appears to be an important growth and survival factor for myeloma cells and confers resistance to treatment with
Bisphosphonate structures dexamethasone, a commonly used treatment for multiple myeloma.
Metalloproteinases (MMPs), important for normal and malignant remodelling, may also contribute. Bone marrow stromal cells from myeloma patients secrete interstitial collagenases (MMP-1) and gelatinase A (MMP-2). MMP-1 initiates bone resorption by degrading type I collagen, which becomes a substrate for MMP-2. Malignant plasma
cells have been found to upregulate MMP-1 and activate MMP-2.

In myeloma osteoprotegerin (OPG) has recently been found to be a survival factor for myeloma cells in vitro (Shipman & Croucher, 2003). Tumour Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL/Apo2L), also a member of the TNF receptor family, selectively clodronate induces apoptosis of tumour cells in vitro whilst not affecting normal cells. However, Shipman and Croucher found that recombinant OPG and natural OPG produced by osteoblast-like cells significantly reduced TRAIL/Apo2L-induced apoptosis of myeloma cells in vitro. This implies
that OPG may act as a survival factor for myeloma, though how this translates to the in vivo setting is not yet clear as it is unknown how OPG and TRAIL/Apo2L interacts within the human bone microenvironment.

Pharmacology of Bisphosphonates
Bisphosphonates are stable analogues of pyrophosphate (PPi).
Pyrophosphate has a P-O-P structure (Figure 2), whereby two phosphate groups are linked by an oxygen atom. Bisphosphonates, however, have a P-C-P structure, a geminal (central) carbon atom replacing the oxygen.
Side chains R1 and R2 are attached to the carbon atom and influence the bisphosphonates’ ability to bind to bone, and their anti-resorptive ability. Bisphosphonates containing a primary nitrogen atom in the R2 side-chain (for example pamidronate) are more potent than non-nitrogen bisphosphonates, (such as clodronate), whilst modifying the primary amine to form a tertiary amine (for example ibandronate) results in even more potent molecules. The most potent bisphosphonates to date appear to be those containing a tertiary amine within a ring structure, such as zoledronic acid.

Bisphosphonates bind avidly to hydroxyapaptite bone mineral surfaces and are selectively internalised by osteoclasts where they inhibit their activity (Russell & Rogers, 1999) (Figure 3). Nitrogen-containing
Cells bisphosphonates inhibit the mevalonate pathway, the main target being farnesyl diphosphate (FPP) synthase. (Dunford et al., 2001). Inhibition of the mevalonate pathway leads to loss of important prenylated proteins which are required for post-translation lipid modification (that is, prenylation) of signalling GTPases, such as Ras, Rho and Rac.

These regulate a variety of key osteoclast cell functions such as control of endosomes, integrin signalling, membrane ruffling, control of cell morphology, and loss of these proteins leads to induction of osteoclast apoptosis. Non nitrogen-containing bisphosphonates have a different mechanism of action. They are metabolically incorporated into nonhydrolysable analogues of ATP, ultimately also leading to osteoclast apoptosis. As a consequence of this osteolysis is effectively inhibited.

Anti-tumour Effects of Bisphosphonates
Increasing evidence is accumulating that bisphosphonates are able to directly affect tumour cells, in addition to their direct effects upon steoclasts. Potency of anti-tumour effect in vitro generally mirrors potency of anti-resorptive ability with nitrogen-bisphosphonates, in particular, zoledronic acid, being the most potent in both respects. Bisphosphonates induce apoptosis of tumour cells and inhibit tumour cell growth, in vitro, of a variety of tumour cell types, including: breast, prostate, melanoma, osteosarcoma, and myeloma, (Jagdev et al., 2001a, Neville-Webbe et al. 2002) tumour cells. The mechanism of apoptosis, at least for breast and myeloma cells, appears to be through inhibition of the mevalonate pathway for nitrogen-bisphosphonates, as for induction of osteoclast apoptosis. (Jagdev et al., 2001a; Shipman et al., 1998).

Nitrogen-bisphosphonates inhibit adhesion and spreading of human breast, prostate, fibrosarcoma cell lines and human melanoma cell lines to and over bone matrix, in vitro, (van der Pluijm et al., 1996), (Boissier et al., 2000) whereas non nitrogen-bisphosphonates have little or no effect. Bisphosphonates also inhibit various MMPs, such as MMP 2,9, and 12, which are particularly involved in cancer growth and metastases in vitro. (Boissier et al., 2000). Interestingly for this particular anti-tumour activity, all bisphosphonates appeared equi-potent. Consequently bisphosphonates are able to inhibit invasion of breast and prostate cancer cells though artificial membrane in vitro. This is thought to be due to bisphosphonates effects upon MMPs, but recent data reveal, for zoledronic acid at least, that inhibition of invasion may also be due to the effects of zoledronic acid on the mevalonate pathway.

More recently, possible anti-angiogenic effects of bisphosphonates have been discovered, neo-angiogenesis being a prerequisite for cancer cell growth and spread. Osteoblastic cells in the bone marrow produce both vascular endothelial growth factor (VEGF) and basic fibroblastic growth factor (b-FGF) and vascularisation is needed for osteoclastic bone resorption. Intravenous zoledronic acid (4 mg), or pamidronate (90 mg), given as treatment for metastatic bone disease for breast cancer patients, induced a significant decrease in bone marrow plasma values of b-FGF and in VEGF, three days post infusion (Jagdev et al., 2001b).

Additionally, Santini et al (Santini et al., 2002) found that pamidronate was able to induce significant decreases in serum VEGF of cancer patients with a variety of solid tumours (including NSCLC, breast, prostate and bladder cancers) that had spread to bone. Significant decreases in serum VEGF level were found 24 hours post-90 mg infusion of pamidronate, with further significant decreases at two days. Zoledronic acid and pamidronate have also been shown to decrease b-FGF and to a lesser extent, VEGF-induced proliferation of vascular tissue in a murine soft tissue model of angiogenesis (Wood et al., 2002). This indicates that nitrogenbisphosphonates may have anti-angiogenic potential outside the bone microenvironment, with zoledronic acid the more potent inhibitor of the two compounds on angiogenesis.

Of particular interest is the potential for bisphosphonates, when used in vitro, to enhance the anti-tumour activity of known cytotoxic agents that are commonly used in the clinical setting (Figure 3). Whilst there are data derived from pre-clinical experiments exploring potential interactions of various cytotoxic drugs in use for particular tumour types, very little is known about how bisphosphonates may interact with commonly used cytotoxic agents. This is especially pertinent for metastatic breast cancer, as these patients are often managed with a variety of drugs that may well include a bisphosphonates with an anti-hormonal agent or chemotherapy drug. To date only zoledronic acid has shown synergy in vitro —with paclitaxel or tamoxifen in breast cancer (Jagdev et al., 2001a), and with dexamethasone in myeloma. Other bisphosphonates in combination with cytotoxic drugs, have shown additive activity at best, for example ibandronate in combination with the taxanes in breast cancer, or no effect, for example pamidronate in combination with the chemotherapy agent dacarbazine (Neville-Webbe et al., 2002).

The clinical relevance of the anti-tumour effects of bisphosphonates observed in vitro is not yet clear, though attempts have been made to mimic the human situation of metastatic disease using specially designed animal models of bone metastases. One such example is the 4T1 orthotopic murine model, (Mundy et al., 2001). In this model, murine mammary tumour cells are injected into the mammary fat pad, and subsequent disease progression mimics the human situation. The primary tumour develops by week 1, liver and lung metastases at week 2, bone and other metastases by week 3, and death by weeks 4 or 5. Using this model zoledronic acid, given as a single 0.3µg intravenous injection on day 7 (i.e. the primary tumour had developed), not only led to a reduction inbone lesions and osteolysis, but interestingly also caused a decrease in bone tumour burden. Non-osseous tumour was not affected however. Other bisphosphonates in similar animal models have either shown no effect, or an adverse effect (Neville-Webbe et al., 2002).

In a model of breast cancer bone metastases risedronate, a nitrogen-containing bisphosphonates, was administered at a dose of 4 µg/animal/ day, by subcutaneous injection. This was started either after osteolytic lesions had developed (day 17) for 10 days, continuously for 28 days from the point of tumour cell inoculation, or 7 days before tumour cell injection (Sasaki et al., 1995). In all three groups risedronate inhibited the development of bone metastases, and for those mice receiving continuous risedronate, survival was increased compared to untreated mice. However, whilst risedronate-treated animals had a significant decrease in bone tumour-load, there was a greater amount of metastatic invasion into soft tissues surrounding bone when compared with the untreated animals. In contrast, the experimental bisphosphonate YH529, when started the same day as MDA-MB-231 breast cancer cell inoculation into nude mice, (20 µg/day/s.c. for 4 weeks) reduced non-osseous metastases, in addition to the inhibition of bone lesions and tumour burden in bone (Sasaki et al., 1998).

Collating data accrued from pre-clinical studies suggest that bisphosphonates not only affect osteoclasts in the bone, but also appear to directly affect tumour cells, albeit within the bone microenvironment. Bisphosphonates may induce tumour cell death directly, for example by induction of apoptosis, or inhibition of their growth and spread, or indirectly by cutting-off their 'survival factors’ such as transforming growth factor beta (TGF-â) and insulin-like growth factor-1 (IGF-1), which are released from the resorbing bone (Figure 4). The clinical relevance of these observations is under investigation.

References

1. Boissier S, Ferreras M, Peyruchaud O, Magnetto S, Ebetino FH, Colombel M, Delmas P, Delaisse JM, Clezardin P Bisphosphonates inhibit breast and prostate carcinoma cell invasion, an early event in the formation of bone metastases Cancer Res 2000;60:2949-2954.
2. Bryden AA, Hoyland JA, Freemont AJ, Clarke NW, George NJ Parathyroid hormone related peptide and receptor expression in paired primary prostate cancer and bone metastases Br J Cancer 2002;86: 322-325.
3. Dunford JE, Thompson K, Coxon FP, Luckman SP, Hahn FM, Poulter CD, Ebetino FH, Rogers MJ Structure-activity relationships for inhibition of farnesyl diphosphate synthase in vitro and inhibition of bone resorption in vivo by nitrogen-containing bisphosphonates J Pharmacol Exp Ther 2001;296:235-242.
4. Garnero P, Buchs N, Zekri J, Rizzoli R, Coleman RE, Delmas, PD Markers of bone turnover for the management of patients with bone metastases from prostate cancer Br J Cancer 2000;82:858-864.
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7. Jagdev SP, Coleman RE, Shipman CM, Rostami HA, Croucher PI The bisphosphonate, zoledronic acid, induces apoptosis of breast cancer cells: evidence for synergy with paclitaxel Br J Cancer 2001a;84: 1126-1134.
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19. Van der Pluijm G, Vloedgraven H, van Beek E, van der Wee-Pals L, Lowik C, Papapoulos S Bisphosponates inhibit the adhesion of breast cancer cells to bone matrices in vitro. J Clin Invest 1996;98:698-705.
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