Julie M. Blair

Mechanisms of Disease: roles of OPG, RANKL and RANK in the pathophysiology of skeletal metastasis

The discovery of osteoprotegerin, receptor activator of nuclear factor kappa B (RANK) and RANK ligand as critical molecular determinants of osteoclastogenesis and regulators of bone resorption, has revolutionized our understanding of the processes of normal and pathological bone biology. Altering the relative biological availabilities of these molecules has direct consequences for the regulation of both bone resorption and bone remodeling. Importantly, recent research suggests a pivotal role for these molecules in mediating cancer-induced bone destruction. This review summarizes the current evidence of osteoprotegerin, RANK ligand and RANK involvement in the pathophysiology of skeletal metastasis, and of therapeutic targeting of this process.

Osteoprotegerin in Prostate Cancer Bone Metastasis

Osteoprotegerin (OPG), a critical regulator of osteoclastogenesis, is expressed by prostate cancer cells, and OPG levels are increased in patients with prostate cancer bone metastases. The objective of this study was to investigate the effects of OPG overexpression on prostate cancer cells and prostate cancer/bone cell interactions in vitro and in vivo. OPG-transfected C4-2 cells expressed 8.0 ng OPG per mL per 10(6) cells, whereas no OPG was detected in the media of C4-2 cells transfected with a control plasmid. OPG overexpressed by C4-2 cells protected these cells from tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis and decreased osteoclast formation. Subcutaneous OPG-C4-2 and pcDNA-C4-2 tumors exhibited similar growth and take-rate characteristics. However, when grown in bone, tumor volume was decreased in OPG-C4-2 versus pcDNA-C4-2 (P=0.0017). OPG expressed by C4-2 cells caused increases in bone mineral density (P=0.0074) and percentage of trabecular bone volume (P=0.007), and decreases in numbers of osteoblasts and osteoclasts when compared with intratibial pcDNA-C4-2 tumors (P=0.003 and P=0.019, respectively). In summary, our data show that increased expression of OPG in C4-2 cells does not directly affect proliferation of prostate cancer cells but indirectly decreases growth of C4-2 tumors in the bone environment. Our data also show that OPG expressed by C4-2 cells inhibits bone lysis associated with C4-2 bone metastasis, which results in net increases in bone volume. We therefore hypothesize that OPG expressed in prostate cancer patient bone metastases may be at least partially responsible for the osteoblastic character of most prostate cancer bone lesions.

Prolonged antiresorptive treatment of lytic prostate cancer xenografts in mouse bone results in tumor necrosis

AIM: In this study, we examine the time dependence of commencing antiresorptive treatments, using an Fc-fusion osteoprotegerin construct (Fc-OPG), on the growth, viability and lytic effects of PC3 human prostate cancer xenografts in male mouse bone. METHODS: Male mice were implanted with PC3 cells by intratibial injection. Mice were randomized into three groups (n=5-7 per group) to receive vehicle or 3 mg/kg Fc-OPG three times weekly from the week before cell injection (week-1; early treatment), week 0 (simultaneous treatment) or week 2 (delayed treatment). RESULTS: When compared with vehicle-treated controls, Fc-OPG treatments protected bone and reduced bone turnover marker levels. In early and simultaneous Fc-OPG treated mice, necrosis of bone-enclosed tumor and the adjacent endosteal bone was observed. No tissue or bone necrosis was seen in sham-injected or vehicletreated controls or in the contralateral tibiae of Fc-OPG treated mice. CONCLUSION: The necrosis of bone-confined tumor in Fc-OPG-treated mice suggests that prolonged blockage of bone erosion during the early development of a tumor mass in bone can limit vascular supply, leading to necrosis. The loss of osteocyte viability was only seen adjacent to necrotic tumor, which suggests that this effect is not due to bone resorption inhibition per se but to interactions between inhibition of bone resorption and tumor effects.

Androgen decreases osteoprotegerin expression in prostate cancer cells

Osteoprotegerin (OPG), a key regulator of bone resorption, is hypothesized to have a role in prostate cancer (CaP) bone metastasis. As advanced CaP is treated by androgen ablation, we examined if androgen modulates OPG expression by CaP cell lines in vitro. Basal levels of secreted OPG protein were significantly greater in androgen-independent PC-3 cells compared with androgen-responsive LNCaP-FGC cells (P<0.001); OPG was not detected in the androgen-responsive CaP cell lines LAPC-4 or DuCaP. Treatment with 5α-dihydrotestosterone (5α-DHT) significantly decreased OPG protein levels in both PC-3 and LNCaP-FGC, with maximal suppression using 10−9–10−7 M 5α-DHT in PC-3 (P<0.01; day 3), and using 10−10–10−9 M 5α-DHT in LNCaP-FGC cells (P<0.01; day 6). OPG messenger RNA levels were not significantly altered by this 5α-DHT treatment. Co-treatment with 10−6 M flutamide blocked 5α-DHT inhibition of OPG protein expression in LNCaP-FGC cells. These data suggest that androgen may modulate OPG protein levels in CaP cells lines in vitro using a post-transcriptional mechanism.

Bone, Mineral, Connective Tissue Metabolism

Metabolic bone diseases result from many different pathological processes which have in common a deleterious effect on bone architecture and strength. The number of people worldwide affected by metabolic bone diseases is substantial and increasing with the aging of many populations. The most common is osteoporosis, which is characterized by reduced bone mineral density and increased bone fragility. It has been estimated that more than 1.3 million osteoporotic fractures/year occur in the USA alone. The fractures associated with osteoporosis and the skeletal complications of arthritis, cancer metastases, and other bone diseases together produce significant morbidity and mortality in those affected, and are associated with enormous cost to health providers. There is significant unmet medical need for new therapies to supplement the currently approved drugs. This chapter reviews the normal physiological regulation of bone remodeling and describes how dysregulation of these processes leads to bone disease. The major bone diseases are described and the current treatments outlined. The strategies required for development of new treatments to meet the continuing unmet medical need are discussed.