Osteoprotegerin overexpression by breast cancer cells enhances orthotopic and osseous tumor growth and contrasts with that delivered therapeutically.

Osteoprotegerin (OPG) acts as a decoy receptor for receptor activator of NF-kappaB ligand (RANKL), which is a pivotal molecule required for osteoclast formation. In vitro OPG inhibits osteoclast formation and in vivo (administered as Fc-OPG) it reduces hypercalcemia and the establishment of osteolytic lesions in mouse models of tumor cell growth in bone. Osteolysis can be induced by parathyroid hormone-related protein (PTHrP) produced by breast cancer cells that results in an increased osteoblastic RANKL/OPG ratio. We examined the effect of local tumor production of OPG on the ability of breast cancer cells to establish and grow in bone and mammary fat pad. MCF-7 cells or MCF-7 cells overexpressing PTHrP were transfected with full-length OPG and inoculated into the proximal tibiae of athymic nude mice. Mice injected with cells overexpressing PTHrP and OPG showed enhanced tumor growth, increased osteolysis (2-fold compared with MCF-7 cells overexpressing PTHrP), and altered histology that was reflective of a less differentiated (more aggressive) phenotype compared with MCF-7 cells. In contrast, administration of recombinant Fc-OPG reduced tumor growth and limited osteolysis even in mice inoculated with OPG overexpressing cells. Similarly, OPG overexpression by breast cancer cells enhanced tumor growth following orthotopic inoculation. These results indicate that OPG overexpression by breast cancer cells increases tumor growth in vivo and that there are strikingly different responses between therapeutically administered Fc-OPG and full-length OPG produced by tumor cells.

Tumor-specific expression of alphavbeta3 integrin promotes spontaneous metastasis of breast cancer to bone.

INTRODUCTION: Studies in xenograft models and experimental models of metastasis have implicated several beta3 integrin-expressing cell populations, including endothelium, platelets and osteoclasts, in breast tumor progression. Since orthotopic human xenograft models of breast cancer are poorly metastatic to bone and experimental models bypass the formation of a primary tumor, however, the precise contribution of tumor-specific alphavbeta3 to the spontaneous metastasis of breast tumors from the mammary gland to bone remains unclear. METHODS: We used a syngeneic orthotopic model of spontaneous breast cancer metastasis to test whether exogenous expression of alphavbeta3 in a mammary carcinoma line (66cl4) that metastasizes to the lung, but not to bone, was sufficient to promote its spontaneous metastasis to bone from the mammary gland. The tumor burden in the spine and the lung following inoculation of alphavbeta3-expressing 66cl4 (66cl4beta3) tumor cells or control 66cl4pBabe into the mammary gland was analyzed by real-time quantitative PCR. The ability of these cells to grow and form osteolytic lesions in bone was determined by histology and tartrate-resistant acid phosphatase staining of bone sections following intratibial injection of tumor cells. The adhesive, migratory and invasive properties of 66cl4pBabe and 66cl4beta3 cells were evaluated in standard in vitro assays. RESULTS: The 66cl4beta3 tumors showed a 20-fold increase in metastatic burden in the spine compared with 66cl4pBabe. A similar trend in lung metastasis was observed. alphavbeta3 did not increase the proliferation of 66cl4 cells in vitro or in the mammary gland in vivo. Similarly, alphavbeta3 is not required for the proliferation of 66cl4 cells in bone as both 66cl4pBabe and 66cl4beta3 proliferated to the same extent when injected directly into the tibia. 66cl4beta3 tumor growth in the tibia, however, increased osteoclast recruitment and bone resorption compared with 66cl4 tumors. Moreover, alphavbeta3 increased 66cl4 tumor cell adhesion and alphavbeta3-dependent haptotactic migration towards bone matrix proteins, as well as their chemotactic response to bone-derived soluble factors in vitro. CONCLUSION: These results demonstrate for the first time that tumor-specific alphavbeta3 contributes to spontaneous metastasis of breast tumors to bone and suggest a critical role for this receptor in mediating chemotactic and haptotactic migration towards bone factors.

Osteoprotegerin reduces osteoclast numbers and prevents bone erosion in collagen-induced arthritis.

Rheumatoid arthritis is characterized by progressive synovial inflammation and joint destruction. While matrix metalloproteinases (MMPs) are implicated in the erosion of unmineralized cartilage, bone destruction involves osteoclasts, the specialized cells that resorb calcified bone matrix. RANK ligand (RANKL) expressed by stromal cells and T cells, and its cognate receptor, RANK, were identified as a critical ligand-receptor pair for osteoclast differentiation and survival. A decoy receptor for RANKL, osteoprotegerin, (OPG) impinges on this system and regulates osteoclast numbers and activity. RANKL is also expressed in collagen-induced arthritis (CIA) in which focal collections of osteoclasts are prominent at sites of bone destruction. To determine the role of RANK signaling events in the effector phase of CIA, we investigated effects of Fc-osteoprotegerin fusion protein (Fc-OPG) in CIA. After induction of CIA in Dark Agouti rats, test animals were treated with or without Fc-OPG (3 mg/kg/day) subcutaneously for 5 days, beginning at the onset of disease. Paraffin-embedded joints were then analyzed histologically and the adjacent bone assessed by histomorphometry. Osteoclasts were identified using TRAP staining and expression of the mRNA for OPG and RANKL was identified by in situ hybridization. The results indicated that short-term Fc-OPG effectively prevented joint destruction, even though it had no impact on the inflammatory aspects of CIA. In arthritic joints, Fc-OPG depleted osteoclast numbers by over 75% and diminished bone erosion scores by over 60%. Although cartilage loss was also reduced by Fc-OPG, the effects on cartilage were less striking than those on bone. In arthritic joints OPG mRNA was highly expressed and co-localized with RANK ligand, and treatment with Fc-OPG did not affect the expression of endogenous RANKL or OPG mRNA. These data demonstrate that short term Fc-OPG treatment has powerful anti-erosive effects, principally on bone, even though synovitis is not affected. These findings indicate the potential utility of disrupting RANK signaling to preserve skeletal integrity in inflammatory arthritis.