CCN3 impairs osteoblast and stimulates osteoclast differentiation to favor breast cancer metastasis to bone

Bone is a preferred site for breast cancer metastasis, causing pain, fractures, spinal cord compressions, and hypercalcemia, all of which can significantly diminish the patient's quality of life. We identified CCN3 as a novel factor that is highly expressed in bone metastatic breast cancer cells from a xenograft mouse model and in bone metastatic lesions from patients with breast cancer. We demonstrate that CCN3 overexpression enhances the ability of weakly bone metastatic breast cancer cells to colonize and grow in the bone without altering their growth in the mammary fat pad. We further demonstrated that human recombinant CCN3 inhibits osteoblast differentiation from primary bone marrow cultures, leading to a higher receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) ratio. In conjunction with its ability to impair osteoblast differentiation, we uncovered a novel role for CCN3 in promoting osteoclast differentiation from RANKL-primed monocyte precursors. CCN3 exerts its pro-osteoclastogenic effects by promoting calcium oscillations and nuclear factor of activated T cells c1 (NFATc1) nuclear translocation. Together, these results demonstrate that CCN3 regulates the differentiation of bone resident cells to create a resorptive environment that promotes the formation of osteolytic breast cancer metastases.     

Oestrogen receptor positive breast cancer metastasis to bone: inhibition by targeting the bone microenvironment in vivo

Clinical trials have shown that adjuvant Zoledronic acid (ZOL) reduces the development of bone metastases irrespective of ER status. However, post-menopausal patients show anti-tumour benefit with ZOL whereas pre-menopausal patients do not. Here we have developed in vivo models of spontaneous ER+ve breast cancer metastasis to bone and investigated the effects of ZOL and oestrogen on tumour cell dissemination and growth. ER+ve (MCF7, T47D) or ER?ve (MDA-MB-231) cells were administered by inter-mammary or inter-cardiac injection into female nude mice ± estradiol. Mice were administered saline or 100 μg/kg ZOL weekly. Tumour growth, dissemination of tumour cells in blood, bone and bone turnover were monitored by luciferase imaging, histology, flow cytometry, two-photon microscopy, micro-CT and TRAP/P1NP ELISA. Estradiol induced metastasis of ER+ve cells to bone in 80–100 % of animals whereas bone metastases from ER?ve cells were unaffected. Administration of ZOL had no effect on tumour growth in the fat pad but significantly inhibited dissemination of ER+ve tumour cells to bone and frequency of bone metastasis. Estradiol and ZOL increased bone volume via different mechanisms: Estradiol increased activity of bone forming osteoblasts whereas administration of ZOL to estradiol supplemented mice decreased osteoclast activity and returned osteoblast activity to levels comparable to that of saline treated mice. ER?ve cells require increased osteoclast activity to grow in bone whereas ER+ve cells do not. Zol does not affect ER+ve tumour growth in soft tissue, however, inhibition of bone turnover by ZOL reduced dissemination and growth of ER+ve breast cancer cells in bone.     

Lack of noggin expression by cancer cells is a determinant of the osteoblast response in bone metastases

Prostate and mammary cancer bone metastases can be osteoblastic or osteolytic, but the mechanisms determining these features are unclear. Bone morphogenetic and Wnt proteins are osteoinductive molecules. Their activity is modulated by antagonists such as noggin and dickkopf-1. Differential expression analysis of bone morphogenetic and Wnt protein antagonists in human prostate and mammary cancer cell lines showed that osteolytic cell lines constitutively express in vitro noggin and dickkopf-1 and at least one of the osteolytic cytokines parathyroid hormone-related protein, colony-stimulating factor-1, and interleukin-8. In contrast, osteoinductive cell lines express neither noggin nor dickkopf-1 nor osteolytic cytokines in vitro. The noggin differential expression profile observed in vitro was confirmed in vivo in prostate cancer cell lines xenografted into bone and in clinical samples of bone metastasis. Forced noggin expression in an osteoinductive prostate cancer cell line abolished the osteoblast response induced in vivo by its intraosseous xenografts. Basal bone resorption and tumor growth kinetics were marginally affected. Lack of noggin and possibly dickkopf-1 expression by cancer cells may be a relevant mechanism contributing to the osteoblast response in bone metastases. Concomitant lack of osteolytic cytokines may be permissive of this effect. Noggin is a candidate drug for the adjuvant therapy of bone metastasis.     

Bone metastasis: pathogenesis and therapeutic implications

Advanced cancers are prone to metastasize. Visceral metastases are more likely to be fatal, while patients with only metastases to bone can survive up to 10 years or more. However, effective treatments for bone metastases are not yet available and bisphosphonates improve the quality of life with no life-prolonging benefits. Bone metastases are classified as osteolytic, osteosclerotic or mixed lesions according to the bone cell types more prominently involved. Either conditions induce high morbidity and dramatically increase the risk of pathological fractures. Several molecular mechanisms bring about cancer cells to metastasize to bone, and osteotropic cancer cells are believed to acquire bone cell-like properties which improve homing, adhesion, proliferation and survival in the bone microenvironment. The acquisition of a bone cell pseudo-phenotype, denominated osteomimicry, is likely to rely on expression of osteoblastic and osteoclastic genes, thus requiring a multigenic programme. Several microenvironmental factors improve the ability of cancer cells to develop at skeletal sites, and a reciprocal deleterious stimulation generates a vicious cycle between the tumour cells and the cells residing in the bone environment. The impact of the stem cell niche in the development of bone metastases and in the phenomenon of tumour dormancy, that allows tumour cells to remain quiescent for decades before establishing overt lesions, is at present only speculative. However, the osteoblast niche, known to maintain the haematopoietic stem cell population in a quiescent status, is likely to be involved in the development of bone metastases and this promising research field is rapidly expanding.     

Receptor Activator for Nuclear Factor kappa B Ligand (RANKL) as an osteoimmune key regulator in bone physiology and pathology

The strength and integrity of the human skeleton depends on a delicate equilibrium between bone resorption and bone formation. Bone resorption is an elementary cellular activity in the modelling of the skeleton during growth and development. Later in life a most important physiological process in the skeleton is bone remodelling, which is locally initiated by resorption. During remodelling bone resorption is coupled to new bone formation that ensures renewal of bone with only minor local and temporary bone loss. Cells responsible for bone resorption and subsequent bone formation are the osteoclasts and osteoblasts, respectively. The osteoclast is derived from the pluripotent hematopoietic stem cell, which gives rise to a myeloid stem cell that can further differentiate into megakaryocytes, granulocytes, monocytes/macrophages and osteoclasts. The respective bone resorbing and forming actions of osteoclasts and osteoblasts are finely coupled, so that bone mass remains remarkably stable in a healthy adult. Imbalance between osteoclast and osteoblast activities can arise from a wide variety of hormonal changes or perturbations of inflammatory and growth factors resulting in postmenopausal osteoporosis, Paget's disease, lytic bone metastases, or rheumatoid arthritis, leading to increased bone resorption and crippling bone damage. In view of the critical role of osteoclasts in diverse pathology, there has been immense effort aimed at understanding the biology of this unique cell. The present review is focused on the current knowledge of the mechanisms that regulate the functional links between bone turnover and the immune system helping us to understand the main factors that lead to bone loss observed in osteoporosis, cancer and in rheumatoid arthritis. The aim of this review paper is to consider the key molecular interactions involved in the formation of osteoclast cells in normal and pathological conditions.     

Histomorphometric analysis of osteoclastic bone resorption in metastatic bone disease from various primary malignomas

Summary The present study deals with qualitative und quantitative analysis of osteoclastic bone resorption in metastatic bone disease. 267 cases were examined histomorphologically and divided into three developmental stages. In the first phase of early appearance no bone resorption takes place. The stimulation of osteoclastic resorption in the surroundings of tumour tissue is typical in the second phase of interaction. Pressure atrophy, aseptic necrosis and osteolysis by the tumour cells themselves are other mechanisms of bone destruction in the last phase of carcinomatosis. Because osteoclasts are exclusively responsible for the loss of bone tissue in the phase of interaction, this stage is suited for precise quantitative analysis of osteoclastic resorption. 24 pure osteolytic secondary bone tumours of various primary lesions were examined histomorphometrically. The numerical values were compared with each other and with standard values of healthy individuals. In contrast with normal bone tissue the fractional resorption surfaces und osteoclast indices increase in metastases. Activated osteoclasts are larger and have more nuclei. The numbers of osteoclast index and nuclei per osteoclast are significantly higher in renal than in breast carcinoma. Osteoclasts can be activated in distances of more than 500 µm from tumour tissue. The mean stimulation distance in metastasis from squamous cell carcinoma is markedly higher than in secondary bone tumours of breast carcinoma. Several osteoclast activating substances and divers mechanisms of stimulation might be responsible for different numerical values of morphometric parameters in metastases from various primary malignancies.Dedicated to Prof. Dr. G. Seifert on the occasion of his 65th birthday     

破骨细胞功能调控与骨质疏松症

Osteoclast(OC)are bone resorbing cells whose development and activity are under the influence of osteoblast(OB).The potential association between those two cell lineages is an area of significant interest.It was not until 1998 that osteoclast differentiation factor(ODF)and osteoprotegerin(OPG)was clonedl.The two novel TNF superfamily members have been recently demonstrate to be the key extracellular regulators of osteoclasteogenesis and bone resorption both in vivo and in vitro,and they play important roles in osteoporosis development,diganosis and treatment.     

Inhibition of RANKL blocks skeletal tumor progression and improves survival in a mouse model of breast cancer bone metastasis

Bone metastases cause severe skeletal morbidity including fractures and hypercalcemia. Tumor cells in bone induce activation of osteoclasts, which mediate bone resorption and release of growth factors from bone matrix, resulting in a “vicious cycle” of bone breakdown and tumor proliferation. Receptor activator of NF-κB ligand (RANKL) is an essential mediator of osteoclast formation, function, and survival, and is blocked by a soluble decoy receptor, osteoprotegerin (OPG). In human malignancies that metastasize to bone, dysregulation of the RANK/RANKL/OPG pathway can increase the RANKL:OPG ratio, a condition which favors excessive osteolysis. In a mouse model of bone metastasis, RANKL protein levels in MDA-MB-231 (MDA-231) tumor-bearing bones were significantly higher than tumor-free bones. The resulting tumor-induced osteoclastogenesis and osteolysis was dose-dependently inhibited by recombinant OPG-Fc treatment, supporting the essential role for RANKL in this process. Using bioluminescence imaging in a mouse model of metastasis, we monitored the anti-tumor efficacy of RANKL inhibition on MDA-231 human breast cancer cells in a temporal manner. Treatment with OPG-Fc in vivo inhibited growth of MDA-231 tumor cells in bony sites when given both as a preventative (dosed day 0) and as a therapeutic agent for established bone metastases (dosed day 7). One mechanism by which RANKL inhibition reduced tumor burden appears to be indirect through inhibition of the “vicious cycle” and involved an increase in tumor cell apoptosis, as measured by active caspase-3. Here, we demonstrate for the first time that OPG-Fc treatment of mice with established bone metastases resulted in an overall improvement in survival.     

Constitutive c-fos expression in osteoblastic MC3T3-E1 cells stimulates osteoclast maturation and osteoclastic bone resorption.

The effect of culture supernatants of c-fos-transfected MC3T3-E1 osteoblastic cells on osteoclastic bone resorption was studied. Human c-fos cDNA was integrated in the expression vector pH8, and the cells were transfected using the calcium phosphate precipitation technique. Osteoclastic bone resorption was quantified by the pit formation assay, and the osteoclast maturation from precursor was assessed by the generation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNC). The culture supernatants of MC3T3-E1 transfectants constitutively expressing c-fos gene enhanced osteoclast-like MNC formation from haematopoietic blast cells compared with those of control transfectants (P < 0.01). The culture supernatants also promoted osteoclastic bone resorption: the pit number, 118.7 +/- 38.5, was significantly higher than 19.0 +/- 10.1 of the control (P < 0.05). The absorption area, 12,394 +/- 3145 mm2, was significantly larger than 1646 +/- 314 mm2 of the control (P < 0.05). The culture supernatants also promoted bone resorption by purified chick osteoclasts (P < 0.05). The results show that constitutive expression of c-fos gene in osteoblastic MC3T3-E1 cells stimulates osteoclast maturation and osteoclastic bone resorption by releasing humoral mediator(s).