osteoprotegerin and Neoplasm-Metastasis

More than 2 decades ago, the discovery of osteoprotegerin (OPG) as inhibitor of the receptor of activator of nuclear factor Kb (RANK) ligand (RANKL) revolutionized our understanding of bone biology and oncology. Besides acting as decoy receptor for RANKL, OPG acts as decoy receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). OPG, RANKL, and TRAIL are ubiquitously expressed, stimulating per se pivotal signaling cascades implicated in cancer. In the context of cancer cell-bone cell interactions, cancer cells skew the OPG/RANKL/RANK (RANKL cognate receptor) balance towards bone destruction and tumor growth through favoring the RANKL/RANK interface, circumventing OPG. Numerous preclinical and clinical studies demonstrate the dual role of OPG in cancer: antitumor and tumor-promoting. OPG potentially conveys an antitumor signal through inhibiting the tumor-promoting RANKL signaling-both the osteoclast-dependent and the osteoclast-independent-and the tumor-promoting TRAIL signaling. On the other hand, the presumed tumor-promoting functions of OPG are: (i) abrogation of TRAIL-induced apoptosis of cancer cells; (ii) abrogation of RANKL-induced antitumor immunity; and (iii) stimulation of oncogenic and prometastatic signaling cascades downstream of the interaction of OPG with diverse proteins. The present review dissects the role of OPG in bone oncology. It presents the available preclinical and clinical data sustaining the dual role of OPG in cancer and focuses on the imbalanced RANKL/RANK/OPG interplay in the landmark "vicious cycle" of skeletal metastatic disease, osteosarcoma, and multiple myeloma. Finally, current challenges and future perspectives in exploiting OPG signaling in bone oncology therapeutics are discussed.

Topics: Animals; Antineoplastic Agents; Apoptosis; Bone and Bones; Bone Neoplasms; Clinical Trials as Topic; Disease Models, Animal; Drug Screening Assays, Antitumor; Humans; Immunoconjugates; Neoplasm Metastasis; Osteoblasts; Osteoclasts; Osteolysis; Osteoprotegerin; RANK Ligand; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand

Bone metastases associated with breast cancer remain a clinical challenge due to their associated morbidity, limited therapeutic intervention and lack of prognostic markers. With a continually evolving understanding of bone biology and its dynamic microenvironment, many potential new targets have been proposed. In this chapter, we discuss the roles of well-established bone markers and how their targeting, in addition to tumour-targeted therapies, might help in the prevention and treatment of bone metastases. There are a vast number of bone markers, of which one of the best-known families is the bone morphogenetic proteins (BMPs). This chapter focuses on their role in breast cancer-associated bone metastases, associated signalling pathways and the possibilities for potential therapeutic intervention. In addition, this chapter provides an update on the role receptor activator of nuclear factor-κB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG) play on breast cancer development and their subsequent influence during the homing and establishment of breast cancer-associated bone metastases. Beyond the well-established bone molecules, this chapter also explores the role of other potential factors such as activated leukocyte cell adhesion molecule (ALCAM) and its potential impact on breast cancer cells' affinity for the bone environment, which implies that ALCAM could be a promising therapeutic target.

Topics: Biomarkers, Tumor; Bone Morphogenetic Proteins; Bone Neoplasms; Breast Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Metastasis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Signal Transduction

Osteoprotegerin (OPG) is a soluble decoy receptor for tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL). It belongs to the tumor necrosis factor receptor superfamily (TNFRSF). OPG was initially discovered to contribute to homeostasis of bone turnover due to its capability of binding to receptor activator of nuclear factor-kappaB (NF-kB). However, apart from bone turnover, OPG plays important and diverse role(s) in many biological functions. Besides having anti-osteoclastic activity, OPG is thought to exert a protective anti-apoptotic action in OPG-expressing tumors by overcoming the physiologic mechanism of tumor surveillance exerted by TRAIL. Along with inhibiting TRAIL induced apoptosis, it can induce proliferation by binding to various cell surface receptors and thus turning on the canonical cell survival and proliferative pathways. OPG also induces angiogenesis, one of the hallmarks of cancer, thus facilitating tumor growth. Recently, the understanding of OPG and its different roles has been augmented substantially. This review is aimed at providing a very informative overview as to how OPG affects cancer progression especially breast cancer.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Breast Neoplasms; Cell Membrane; Cyclooxygenase 2; Humans; Mice; Neoplasm Metastasis; Neovascularization, Pathologic; NF-kappa B; Osteoprotegerin; Polymorphism, Genetic; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; TNF-Related Apoptosis-Inducing Ligand; Treatment Outcome; Tumor Microenvironment

Oncogenic events combined with a favourable environment are the two main factors in the oncological process. The tumour microenvironment is composed of a complex, interconnected network of protagonists, including soluble factors such as cytokines, extracellular matrix components, interacting with fibroblasts, endothelial cells, immune cells and various specific cell types depending on the location of the cancer cells (e.g. pulmonary epithelium, osteoblasts). This diversity defines specific "niches" (e.g. vascular, immune, bone niches) involved in tumour growth and the metastatic process. These actors communicate together by direct intercellular communications and/or in an autocrine/paracrine/endocrine manner involving cytokines and growth factors. Among these glycoproteins, RANKL (receptor activator nuclear factor-κB ligand) and its receptor RANK (receptor activator nuclear factor), members of the TNF and TNFR superfamilies, have stimulated the interest of the scientific community. RANK is frequently expressed by cancer cells in contrast with RANKL which is frequently detected in the tumour microenvironment and together they participate in every step in cancer development. Their activities are markedly regulated by osteoprotegerin (OPG, a soluble decoy receptor) and its ligands, and by LGR4, a membrane receptor able to bind RANKL. The aim of the present review is to provide an overview of the functional implication of the RANK/RANKL system in cancer development, and to underline the most recent clinical studies.

Topics: Clinical Trials as Topic; Disease Progression; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Metastasis; Neoplasms; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, G-Protein-Coupled; Signal Transduction; Tumor Microenvironment

Bone metastasis leads to skeletal-related events in final-stage cancer patients. The incidence of prostate and lung cancers increases yearly; these cancers readily invade the bone. Some recent studies have found that serum osteoprotegerin (OPG) levels may be altered in patients with bone metastasis, whereas other reports have produced inconsistent findings. Hence, we conducted a meta-analysis to examine the effects of lung and prostate cancer on serum OPG levels. A systematic literature search was conducted using PubMed, Medline, and CNKI to identify relevant studies. A total of 11 studies were included. The standardized mean difference (SMD) and 95% confidence interval (95% CI) of the bone metastasis (BM) group, the non-bone metastasis (BM-) group and healthy controls were calculated. In prostate cancer, serum OPG levels in the BM group were higher than in the BM- group and healthy controls. Additionally, no significant difference in serum OPG levels was found between the BM- group and healthy controls. In lung cancer, serum OPG levels in the BM and BM- groups were significantly increased compared with healthy controls. However, no significant difference in serum OPG levels was found between the BM and BM- groups. Studies with larger sample sizes are required to confirm these findings.

Topics: Bone Neoplasms; Humans; Lung Neoplasms; Male; Neoplasm Metastasis; Osteoprotegerin; Prostatic Neoplasms

Cancer development is complex and involves several layers of interactions and pleotropic signaling mechanisms leading to progression. Cancer cells associate with resident stromal fibroblasts, smooth muscle cells, macrophages, endothelium, neurons and migrating cells at metastatic sites and phenotypically and genotypically activate them. These become an integral part of the cancer cell community through activated cell signaling mechanisms. During this process, the cancer cells and cells in the cancer microenvironment "co-evolve" in part due to oxidative stress, and acquire the ability to mimic other cell types (which can be termed osteomimicry, vasculomimicry, neuromimicry and stem cell mimicry), and undergo transition from epithelium to mesenchyme with definitive morphologic and behavioral modifications. In our laboratory, we demonstrated that prostate cancer cells co-evolve in their genotypic and phenotypic characters with stroma and acquire osteomimetic properties allowing them to proliferate and survive in the skeleton as bone metastasis. Several signaling interactions in the bone microenvironment, mediated by reactive oxygen species, soluble and membrane bound factors, such as superoxide, beta2-microglobulin and RANKL have been described. Targeting the signaling pathways in the cancer-associated stromal microenvironment in combination with known conventional therapeutic modalities could have a synergistic effect on cancer treatment. Since cancer cells are constantly interacting and acquiring adaptive and survival changes primarily directed by their microenvironment, it is imperative to delineate these interactions and co-target both cancer and stroma to improve the treatment and overall survival of cancer patients.

Topics: Bone Neoplasms; Disease Progression; Humans; Male; Neoplasm Metastasis; Osteoblasts; Osteoprotegerin; Oxidative Stress; Prostatic Neoplasms; RANK Ligand; Stromal Cells

Osteoprotegerin (OPG), member of tumor necrosis factor (TNF) receptor superfamily, has various biological functions including bone remodeling. OPG binds to receptor activator of nuclear factor-kB ligand (RANKL) and prevents osteoclastic bone resorption. Recently, OPG has gained more clinical interest as its role in cancer-mediated bone destruction and the potential of RANKL inhibition could act as a novel treatment in tumor-induced bone disease. OPG protects prostate cancer cells from apoptotic effects of TRAIL and therefore provides tumor cells producing OPG with survival advantages. Additionally, the increased RANKL/OPG ratio in metastatic breast cancer results in severe osteolysis. Thus, bone formation and resorption are the crux of cancer metastasis, resulting in bone pain and pathological fractures. This review provides an overview of the role of OPG in cancer-induced bone disease.

Topics: Animals; Bone Neoplasms; Bone Resorption; Cell Survival; Humans; Neoplasm Metastasis; Osteoprotegerin; RANK Ligand

Bone metastasis is often the penultimate harbinger of death for many cancer patients. Bone metastases are often associated with fractures and severe pain resulting in decreased quality of life. Accordingly, effective therapies to inhibit the development or progression of bone metastases will have important clinical benefits. To achieve this goal understanding the mechanisms through which bone metastases develop and progress may provide targets to inhibit the metastases. In the past few years, there have been advances in both understanding the mechanisms through which bone metastases develop and how they impact bone remodeling. Additionally, gains in promising clinical strategies to target bone metastases have been developed. In this prospectus, we will discuss some of these advances.

Topics: Antineoplastic Agents; Bone Neoplasms; Breast Neoplasms; Diphosphonates; Female; Humans; Male; Neoplasm Metastasis; Osteolysis; Osteoprotegerin; Prostatic Neoplasms; RANK Ligand

Topics: Animals; Antineoplastic Agents; Bone Neoplasms; Bone Resorption; Carrier Proteins; Diphosphonates; Glycoproteins; Humans; Membrane Glycoproteins; Models, Biological; Neoplasm Metastasis; Osteoclasts; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

The current review summarizes the roles of the ligand, receptor activator of nuclear factor-kappaB ligand (RANKL), its receptor, receptor activator of nuclear factor-kappaB (RANK), and its decoy receptor, osteoprotegerin (OPG), on osteoclast biology and bone resorption. Furthermore, it highlights the impact of these compounds on the pathogenesis of malignant bone diseases, including tumor metastasis, humoral hypercalcemia of malignancy, and multiple myeloma. Finally, the authors discuss the therapeutic potential of OPG in the management of malignancies involving the skeleton.. After its discovery and cloning, the biologic effects of RANKL, RANK, and OPG have been characterized by in vitro experiments and in vivo studies. The generation of knock-out mice and transgenic mice has produced animal models with absent or excessive production of these cytokine components that display opposite abnormal skeletal phenotypes (osteoporosis or osteopetrosis). The potential effect of RANKL and OPG has been assessed by evaluating these compounds in various animal models of metabolic and malignant bone disease and by administering OPG to humans.. Abnormal bone resorption due to local or systemic stimulation of osteoclast differentiation and activation is a hallmark of various benign and malignant bone diseases. RANKL, RANK, and OPG form an essential cytokine system that is capable of regulating all aspects of osteoclast functions, including proliferation, differentiation, fusion, activation, and apoptosis. The balance of bone resorption depends on the local RANKL-to-OPG ratio, which is enhanced in bone metastases and humoral hypercalcemia of malignancy. The exogenous administration of OPG to tumor-bearing animals corrects the increased RANKL-to-OPG ratio, and reverses the skeletal complications of malignancies.. Abnormalities of the RANKL/OPG system have been implicated in the pathogenesis of various primary and secondary bone malignancies. The systemic administration of OPG appears to be a potent novel therapeutic agent for treatment of these disorders.

Topics: Antineoplastic Agents; Biomarkers, Tumor; Bone Diseases; Bone Neoplasms; Carrier Proteins; Glycoproteins; Humans; Membrane Glycoproteins; Neoplasm Metastasis; Osteoprotegerin; Prognosis; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Aberrant activation of fibroblast growth factor receptor (FGFR) signalling contributes to progression and metastasis of many types of cancers including breast cancer. Accordingly, FGFR targeted tyrosine kinase inhibitors (TKIs) are currently under development. However, the efficacy of FGFR TKIs in the bone microenvironment where breast cancer cells most frequently metastasize and also where FGFR is biologically active, has not been clearly investigated. We investigated the FGFR-mediated interactions among cancer and the bone microenvironment stromal cells (osteoblasts and osteoclasts), and also the effects of FGFR inhibition in bone metastasis. We showed that addition of culture supernatant from the MDA-MB-134-VI FGFR-amplified breast cancer cells-activated FGFR siganalling in osteoblasts, including increased expression of RANKL, M-CSF, and osteoprotegerin (OPG). Further in vitro analyses showed that AZD4547, an FGFR TKI currently in clinical trials for breast cancer, decreased RANKL and M-CSF, and subsequently RANKL and M-CSF-dependent osteoclastogenesis of murine bone marrow monocytes. Moreover, AZD4547 suppressed osteoclastogenesis and tumor-induced osteolysis in an orthotopic breast cancer bone metastasis mouse model using FGFR non-amplified MDA-MB-231 cells. Collectively, our results support that FGFR inhibitors inhibit the bone microenvironment stromal cells including osteoblasts and osteoclasts, and effectively suppress both tumor and stromal compartments of bone metastasis.

Topics: Animals; Benzamides; Bone Neoplasms; Breast Neoplasms; Female; Humans; Macrophage Colony-Stimulating Factor; MCF-7 Cells; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Neoplasm Proteins; Osteoclasts; Osteoprotegerin; Piperazines; Pyrazoles; RANK Ligand; Receptor Protein-Tyrosine Kinases; Tumor Microenvironment; Xenograft Model Antitumor Assays

The cytokine system RANKL (receptor activator of NF-κB ligand), its receptor RANK and the antagonist OPG (osteoprotegerin) play a critical role in bone turnover. Our investigation was conducted to describe the gene expression at primary tumour site in prostate cancer patients and correlate the results with Gleason Score and PSA level.. Seventy-one samples were obtained from prostate cancer patients at the time of radical prostatectomy and palliative prostate resection (n = 71). Patients with benign prostate hyperplasia served as controls (n = 60). We performed real-time RT-PCR after microdissection of the samples.. The mRNA expression of RANK was highest in tumour tissue from patients with bone metastases (p < 0.001) as compared to BPH or locally confined tumours, also shown in clinical subgroups distinguished by Gleason Score (< 7 or ≥ 7, p = 0.028) or PSA level (< 10 or ≥ 10 µg/l, p = 0.004). RANKL and OPG mRNA expression was higher in tumour tissue from patients with metastatic compared to local disease. The RANKL/OPG ratio was low in normal prostate tissue and high tumours with bone metastases (p < 0.05). Expression of all three cytokines was high in BPH tissue but did not exceed as much as in the tumour tissue.. We demonstrated that RANK, RANKL and OPG are directly expressed by prostate cancer cells at the primary tumour site and showed a clear correlation with Gleason Score, serum PSA level and advanced disease. In BPH, mRNA expression is also detectable, but RANK expression does not exceed as much as compared to tumour tissue.

Topics: Aged; Aged, 80 and over; Bone Neoplasms; Humans; Kallikreins; Male; Middle Aged; Neoplasm Grading; Neoplasm Metastasis; Osteoprotegerin; Prostate-Specific Antigen; Prostatic Hyperplasia; Prostatic Neoplasms; RANK Ligand; Real-Time Polymerase Chain Reaction; Receptor Activator of Nuclear Factor-kappa B; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcriptome

Osteoprotegerin (OPG) is a soluble receptor of the pro-apoptotic cytokine TRAIL which is thought to contribute to tumour development by inhibiting apoptosis or affecting other aspects of tumour biology, including cell proliferation and immune response. Although immunohistochemical studies suggest that OPG correlates with survival in metastatic colorectal cancer (mCRC), only scarce data are available on serum OPG in CRC patients.. In this pilot study, we assessed the prognostic significance of serum OPG and CEA (Carcinoembryonic antigen) in 81 patients with UICC (Union for International Cancer Control) stage-IV mCRC. OPG was additionally assessed by immunohistochemistry in primary tissue samples from 33 patients of the same cohort.. Baseline serum OPG correlated with CEA (r=0.36, p=0.0011), but independently predicted survival of mCRC patients. Life expectancy was poorer in patients with OPG levels above the median concentration of 51ng/ml (median overall survival [95% confidence interval] 1.8 years [1.3-3.0] vs. 1.0 [0.7-1.2] p=0.013). Patients with high levels of both OPG and CEA had an even poorer life expectancy vs. low-OPG/low-CEA patients (0.9 years [0.6-1.5] vs. 3 years [1.2-4.4], p=0.015), indicating that CEA and OPG have additive prognostic significance. Immunohistochemical analysis of OPG failed to show a correlation between OPG staining and survival (p=0.055) or OPG concentration from matched serum samples.. This pilot study provides evidence of independent prognostic significance of serum OPG in patients with advanced mCRC and warrants its further prospective validation.

Topics: Aged; Biomarkers, Tumor; Carcinoembryonic Antigen; Colorectal Neoplasms; Enzyme-Linked Immunosorbent Assay; Female; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Male; Middle Aged; Neoplasm Metastasis; Neoplasm Staging; Osteoprotegerin; Prognosis; Survival Analysis

We examined the effects of recombinant human osteoclastogenesis inhibitory factor (hOCIF) on osteolysis, proliferation of mammary tumor cells, and induction of cancer stem cells (CSCs) in the tumor-bone and tumor-subcutaneous microenvironments (TB- and TS-microE).. Mouse mammary tumor cells were transplanted onto the calvaria or into a subcutaneous lesion of female mice, creating a TB-microE and a TS-microE, and the mice were then treated with hOCIF. To investigate the preventive effects of hOCIF, mice were treated with hOCIF before tumor cell implantation onto the calvaria (Pre), after (Post), and both before and after (Whole). The number of CSCs and cytokine levels were evaluated by IHC and ELISA assay, respectively.. hOCIF suppressed osteolysis, and growth of mammary tumors in the TB-microE, but not in the TS-microE. In the Pre, Post, and Whole groups, hOCIF suppressed osteolysis, and cell proliferation. hOCIF increased mouse osteoprotegrin (mOPG) levels in vivo, which suppressed mammary tumor cell proliferation in vitro. These preventive effects were observed in the dose-dependent. hOCIF did not affect the induction of CSCs in either microenvironment.. While receptor activator of NF-κB ligand (RANKL) targeting therapy may not affect the induction of CSCs, RANKL is a potential target for prevention as well as treatment of breast cancer bone metastasis.

Topics: Animals; Bone Neoplasms; Cell Line, Tumor; Cell Proliferation; Cytokines; Dose-Response Relationship, Drug; Female; Humans; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neoplastic Stem Cells; Osteolysis; Osteoprotegerin; Recombinant Proteins; Tumor Microenvironment

Osteoprotegrin (OPG), a secreted protein and a member of the tumor necrosis factor receptor superfamily has been well-characterized and is an important regulator of bone remodeling by blocking osteoclast maturation thus preventing osteolysis. In recent years, OPG has been reported to have an association with the malignant capacity of various cancer types and cancer-associated bone metastasis, although the mechanisms of this are not clearly understood.. In this study, OPG expression was analyzed in human lung cancer tissue and normal tissue based on the dataset of The Cancer Genome Atlas and Oncomine. The in vitro effect of OPG on H3122 lung cancer cells was also assessed by characterizing cell function following knock-down and forced overexpression in this cell line.. The expression of OPG was significantly increased in lung cancer tissues compared to the normal control group and OPG promoted the malignant phenotypes of H3122 cells in in vitro models.. OPG may be a potential driver of lung cancer cells and therefore might have potential in therapy and diagnostics.

Topics: Bone Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Lung Neoplasms; Male; Neoplasm Metastasis; Osteoprotegerin

The present study was carried out to investigate the clinical significance of TMPRSS3 and TNFRSF11B in breast cancer. Thus, the expression levels of TMPRSS3 and TNFRSF11B and the correlation with prognosis in patients with breast cancer were analyzed in silico using gene microarray and hierarchical clustering analysis. Then, the differential expression in breast cancer vs. normal breast tissue was explored in the Oncomine platform and verified in our independent samples using IHC technique. Our results indicated that TMPRSS3 was upregulated and TNFRSF11B was downregulated in breast cancer tissues compared with the levels in the human normal breast tissues. TMPRSS3 and TNFRSF11B were confirmed to be correlated with distant organ metastasis of breast cancer. Moreover, upregulation of TMPRSS3 accompanied by downregulation of TNFRSF11B was found to be associated with a shorter median overall survival and indicated a poor prognosis. In conclusion, TMPRSS3 and TNFRSF11B may have potential prognostic value to be used as tumor biomarkers in breast cancer patients.

Topics: Biomarkers, Tumor; Breast Neoplasms; Carcinogenesis; Disease-Free Survival; Female; Gene Expression Regulation, Neoplastic; Humans; Membrane Proteins; Neoplasm Metastasis; Neoplasm Proteins; Osteoprotegerin; Prognosis; Serine Endopeptidases

Osteoprotegrin (OPG), receptor activator of nuclear factor κB (RANK) and RANK ligand (RANKL) are signal transducers which have pleiotropic actions. Each tumour necrosis factor receptor superfamily member has unique structural attributes which directly couples them to signalling pathways involved in cell proliferation, differentiation and survival. Previous studies have clinically linked OPG, RANK and RANKL to increasing tumour burden, metastatic bone involvement and estrogen status. This study aimed to establish the potential implications of targeting endogenously produced OPG and RANK in the osteotropic breast cancer cell line MDA-MB‑231 in vitro. Subsequently this study also aimed to explore the potential links between these molecules with regards to hepatocyte growth factor (HGF) signalling and extracted bone proteins (BME). OPG and RANK expression was successfully suppressed using hammerhead ribozyme technology. Subsequently effects were explored in MDA-MB‑231 cell proliferation, matrix adhesion, migration and invasion in vitro function assays. Reduced OPG expression resulted in increased breast cancer cell migration and invasion. These increases, particularly invasion, appeared to however be reduced under the influence of the exogenous stimuli (HGF and BME). In contrast, suppression of RANK in MDA-MB‑231 breast cancer cells resulted in decreased cancer cell proliferation, matrix-adhesion, motility and invasion with little cumulative effect being noted after the addition of exogenous stimuli. The complexity of the bone environment underpins the vast number of soluble factors and signalling pathways which can influence osteotropic cancer behaviour and progression. Further work into elucidating all the pathways affected could potentially lead to better identification of those patients most at risk.

Topics: Bone and Bones; Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Hepatocyte Growth Factor; Humans; Membrane Glycoproteins; Neoplasm Metastasis; Osteoprotegerin; RANK Ligand; RNA, Catalytic; Signal Transduction

Breast cancer is the most common cause of cancer death among women (522,000 deaths in 2012). Imbalance between RANKL and OPG is observed in many cancers, including breast cancer. Consequently, SNPs in the genes of RANKL and OPG may be involved in breast cancer development. This study included 276 subjects. Group I (n = 100) healthy females as a control group, group II (n = 96) breast cancer patients without bone metastases, and group III (n = 80) breast cancer patients with bone metastases. RANKL rs9533156, OPG rs2073618, and OPG rs2073617 SNPs and their serum protein levels were studied for a possible association with breast cancer development. The allele frequency [(OR: 4.832 CI 2.18-10.71, P = 0.001) and genotype distribution (P = 0.001)] of OPG SNP rs2073618 showed a highly significant difference between breast cancer patients and healthy controls. The allele C is more common in breast cancer patients. The allele frequency [(OR: 0.451 CI 0.232-0.879, P = 0.018) and genotype distribution (P = 0.003)] of RANKL SNP rs9533156 differed significantly between breast cancer patients and healthy controls. The allele T is more common in breast cancer patients. The allele frequency [(OR: 0.36 CI 0.184-0.705, P = 0.002) and genotype distribution (P = 0.011)] of OPG SNP rs2073617 differed significantly between breast cancer patients and healthy controls. The allele T is more common in breast cancer patients. The C allele of OPG SNP rs2073618 may be associated with breast cancer development. No association was found between any of the SNPs and the serum protein levels of RANKL and OPG.

Topics: Breast Neoplasms; Case-Control Studies; Demography; Electrophoresis, Agar Gel; Female; Gene Frequency; Genetic Association Studies; Genetic Predisposition to Disease; Humans; Middle Aged; Neoplasm Metastasis; Osteoprotegerin; Polymorphism, Single Nucleotide; Predictive Value of Tests; RANK Ligand; ROC Curve

Breast cancer frequently spreads to bone. The interaction between bone metastases and microenvironment, referred as the "vicious cycle", increases both tumor burden and bone destruction. Therefore, inhibition at any point in this "vicious cycle" can reduce malignant osteolytic lesions in patients with advanced breast cancer. In this study, we evaluated whether tetrahydrofurofuran-type lignans derived from Magnoliae Flos, commonly used in traditional Asian medicine to treat inflammatory diseases, could block breast cancer-mediated bone loss. Aschatin, fargesin, lirioresinol B dimethyl ether, and magnolin at noncytotoxic concentrations suppressed mRNA expression and secretion of osteolytic factor PTHrP in MDA-MB-231 metastatic human breast cancer cells. Fargesin inhibited TGF-β-stimulated cell viability, migration, and invasion and decreased TGF-β-induced PTHrP production in MDA-MB-231 cells. In addition, these lignans reduced RANKL/OPG ratio in PTHrP-treated hFOB1.19 human osteoblastic cells and inhibited RANKL-mediated osteoclast differentiation in mouse bone marrow macrophages. Aschatin, fargesin, lirioresinol B dimethyl ether, and magnolin substantially reduced bone-resorbing activity of osteoclasts by inhibiting MMP-9 and cathepsin K activities. Furthermore, orally administered fargesin inhibited tumor growth and cancer-mediated bone destruction in mice with MDA-MB-231 cells injected into calvarial tissues. Aschatin, fargesin, lirioresinol B dimethyl ether, and magnolin blocked initiation and progression of the "vicious cycle" between breast cancer metastases and bone microenvironment by inhibiting PTHrP production in breast cancer cells and osteoclastic bone resorption. Therefore, these tetrahydrofurofuran-type lignans have the potential to serve as beneficial agents to prevent and treat cancer-induced bone destruction in breast cancer patients.

Topics: Animals; Benzodioxoles; Bone Resorption; Breast Neoplasms; Cell Line, Tumor; Female; Furans; Gene Expression Regulation, Neoplastic; Humans; Lignans; Lignin; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Osteoblasts; Osteoclasts; Osteoprotegerin; Parathyroid Hormone-Related Protein; RANK Ligand; RNA, Messenger

Osteoprotegerin (OPG) is a secreted member of the tumor necrosis factor (TNF) receptor superfamily that has been well characterized as a negative regulator of bone remodeling. OPG is also expressed in human breast cancer tissues and cell lines. In vitro studies suggest that OPG exerts tumor-promoting effects by binding to TNF-related apoptosis inducing ligand (TRAIL), thereby preventing induction of apoptosis. However, the in vivo effect of OPG expression by primary breast tumors has not been characterized. We knocked down OPG expression in MDA-MB-231 and MDA-MB-436 human breast cancer cells using shRNA and siRNA to investigate impact on metastasis in the chick embryo model. We observed a reduction in metastasis with OPG knockdown cells. We found that lowering OPG expression did not alter sensitivity to TRAIL-induced apoptosis; however, the OPG knockdown cells had a reduced level of invasion. In association with this we observed reduced expression of the proteases Cathepsin D and Matrix Metalloproteinase-2 upon OPG knockdown, indicating that OPG may promote metastasis via modulation of protease expression and invasion. We conclude that OPG has a metastasis-promoting effect in breast cancer cells.

Topics: Animals; Cell Line, Tumor; Cell Survival; Chick Embryo; Disease Models, Animal; Disease Progression; DNA Copy Number Variations; Female; Gene Expression; Gene Knockdown Techniques; Humans; Neoplasm Metastasis; Osteoprotegerin; Patient Outcome Assessment; Peptide Hydrolases; RNA, Messenger; TNF-Related Apoptosis-Inducing Ligand; Triple Negative Breast Neoplasms

Receptor activator of nuclear factor kappa-B and its ligand (RANK/RANKL) and Osteoprotegerin (OPG) are key molecules for regulating osteoclastic activity in bone. However, little is known about the role of RANK-related molecules in breast cancer prognosis. We aimed to evaluate RANK, RANKL, and OPG expression and the associated clinical impact in breast cancer.. Tissue microarray (TMA) from 185 patients with primary breast cancer was established. Immunohistochemistry for RANK, RANKL, and OPG was performed. Clinicopathologic features and survival outcomes associated with expression of RANK, RANKL, and OPG were analyzed.. RANK, RANKL, and OPG were expressed in 74.1%, 78.4%, and 45.9% of patients, respectively. RANKL expression was associated with lower Ki-67 level. OPG expression was related to small tumor size, node negativity, and low Ki-67. There was no significant difference in clinicopathologic features between tumors with RANK and those without RANK. RANK expression was significantly associated with poor disease-free survival in univariate analysis (P = 0.04) and multivariate analysis (P = 0.02). RANKL expression was associated with improved skeletal disease-free survival in multivariate analysis (P = 0.03).. The RANK/RANKL pathway regulated by OPG may have a role in predicting progression and prognosis of breast cancer.

Topics: Biomarkers, Tumor; Breast Neoplasms; Female; Follow-Up Studies; Humans; Immunoenzyme Techniques; Middle Aged; Neoplasm Grading; Neoplasm Metastasis; Neoplasm Recurrence, Local; Osteoprotegerin; Prognosis; Prospective Studies; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Survival Rate

This study is the first to demonstrate that parathyroid hormone-related protein (PTHrP), produced by human breast cancer cells after exposure to phthalate esters, contributes to bone metastasis by increasing osteoclastogenesis. This is also the first to reveal that obtusifolin reverses phthalate esters-mediated bone resorption. Human breast cancer cells were treated with dibutyl phthalate (DBP), harvested in conditioned medium, and cultured to osteoblasts or osteoclasts. Cultures of osteoblasts with DBP-MDA-MB-231-CM increased the osteoclastogenesis activator RANKL (receptor activator of nuclear factor κ-B ligand) and M-CSF (macrophage colony-stimulating factor). PTHrP was secreted in MDA-MB-231 cells. DBP-MDA-MB-231-CM reduced osteoblasts to produce osteoprotegerin, an osteoclastogenesis inhibitor, while DBP mediated PTHrP up-regulation, increasing IL-8 secretion in MDA-MB-231 and contributing to breast cancer-mediated osteoclast differentiation and bone resorption. Obtusifolin, a major bioactive compound present in Cassia tora L., suppressed phthalate esters-mediated bone resorption. Therefore, obtusifolin may be a novel anti-breast-cancer bone metastasis agent.

Topics: Anthraquinones; Bone Neoplasms; Bone Resorption; Cassia; Cell Differentiation; Down-Regulation; Drugs, Chinese Herbal; Esters; Female; Humans; Macrophage Colony-Stimulating Factor; Neoplasm Metastasis; Osteoclasts; Osteoprotegerin; Parathyroid Hormone-Related Protein; Phthalic Acids; RANK Ligand

Bone is a common site of metastasis for lung cancer, and is associated with significant morbidity and a dismal prognosis. MicroRNAs (miRNAs) are increasingly implicated in regulating the progression of malignancies.. The efficacy of miR-33a or anti-miR-33a plasmid was assessed by Real-time PCR. Luciferase assays were using One-Glo Luciferase Assay System. Measurement of secreted factors was determined by ELISA kit.. We have found that miR-33a, which is downregulated in lung cancer cells, directly targets PTHrP (parathyroid hormone-related protein), a potent stimulator of osteoclastic bone resorption, leading to decreased osteolytic bone metastasis. We also found that miR-33a levels are inversely correlated with PTHrP expression between human normal bronchial cell line and lung cancer cell lines. The reintroduction of miR-33a reduces the stimulatory effect of A549 on the production of osteoclastogenesis activator RANKL (receptor activator of nuclear factor kappa-B ligand) and M-CSF (macrophage colony-stimulating factor) on osteoblasts, while the expression of PTHrP is decreased in A549 cells. miR-33a overexpression also reduces the inhibitory activity of A549 on the production of OPG (osteoprotegerin), an osteoclastogenesis inhibitor. In addition, miR-33a-mediated PTHrP downregulation results in decreased IL-8 secretion in A549, which contributes to decreased lung cancer-mediated osteoclast differentiation and bone resorption.. These findings have led us to conclude that miR-33a may be a potent tumor suppressor, which inhibits direct and indirect osteoclastogenesis through repression of PTHrP.. miR-33a may even predict a poor prognosis for lung cancer patients.

Topics: Bone Neoplasms; Bone Resorption; Cell Differentiation; Cell Line, Tumor; Down-Regulation; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Interleukin-8; Lung Neoplasms; MicroRNAs; Neoplasm Metastasis; Neoplasm Proteins; Osteoclasts; Osteoprotegerin; Parathyroid Hormone-Related Protein; RANK Ligand; RNA, Neoplasm

Tumors escape host immune responses, in part, through the release of immunomodulatory factors and decoy receptors into their microenvironment. Several cancers express surface-bound and soluble members of the tumor necrosis factor (TNF) receptor superfamily, including TNFRSF11b/osteoprotegerin (OPG). In its physiologic role, OPG regulates bone remodeling through competition for osteoclast-activating cytokines and protects newly forming bone from T cell-mediated apoptosis. In multiple tumor types, OPG production is associated with an aggressive phenotype and increased metastasis to bone, but no study has examined OPG production in human metastatic melanoma. We demonstrate that a significant proportion of human metastatic melanomas constitutively produces OPG through a mechanism governed by membrane-bound TNF-α signaling through TNF receptor 1 (TNFR1). These observations both define a specific mechanism that regulates melanoma production of OPG and establish a new molecular target for the therapeutic regulation of OPG.

Topics: Apoptosis; Cell Line, Tumor; Cell Membrane; Cytokines; Gene Expression Regulation, Neoplastic; Humans; Immunotherapy; Melanoma; Neoplasm Metastasis; Osteoclasts; Osteoprotegerin; Phenotype; Prognosis; Signal Transduction; Time Factors; Tumor Necrosis Factor-alpha

To determine the mechanism of neuroblastoma (NB) bone invasion/metastasis, it is necessary to investigate the bone invasion/metastasis-related factors in the bone invasion/metastasis process. Some evidence has suggested that various proteins were involved in bone osteolytic response. The invasion/metastasis property and gene expression of NB, however, are still unknown.. Single-cell suspensions of SY5Y and KCNR cells were injected directly into the femur of nude mice. Radiological and histological analyses, immunohistochemistry analyses, and western blot assay were performed to characterize bone metastasis mechanism in these bone metastasis models.. SY5Y and KCNR NB cells result in osteolytic responses in bone metastasis model. Osteoprotegerin (OPG), receptor activator of NF-kappaB ligand (RANKL), parathyroid hormone-related peptide (PTHrP), endothelin 1 (ET-1), and CXCR4 were examined and compared among in vitro, in vivo, and normal bone, respectively. PTHrP, OPG, RANKL, and ET-1 except CXCR4 in SY5Y and KCNR NB cells xenografts were strikingly upregulated compared with normal bone and NB cells. However, significantly stronger expression of PTHrP and RANKL was presented than ET-1 and OPG; furthermore, the ratios of expression of PTHrP, RANKL to OPG, and ET-1 were also markedly increased in vivo versus in vitro.. Our study provided evidence that NB cell may enhance bone invasion through PTHrP, OPG, RANKL, and ET-1, especially PTHrP and RANKL which may display stronger effects. CXCR4 appeared not participating in bone invasion, but in tumor growth, and homing to bone. Targeting PTHrP, OPG, ET-1, and RANKL may provide a new insight and method for patient therapy by inhibiting NB bone metastasis and invasiveness.

Topics: Animals; Blotting, Western; Bone Neoplasms; Cell Line, Tumor; Endothelin-1; Femur; Humans; Immunohistochemistry; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Neuroblastoma; Osteolysis; Osteoprotegerin; Parathyroid Hormone-Related Protein; RANK Ligand; Receptors, CXCR4

In addition to its effects on bone metabolism, osteoprotegerin (OPG), a soluble member of the tumor necrosis factor family of receptors, promotes smooth muscle cell proliferation and migration and may act as a survival factor for tumor cells. We hypothesized that these cellular mechanisms of OPG may be involved in the growth and proliferation of lymphangioleiomyomatosis (LAM) cells, abnormal smooth muscle-like cells with mutations in one of the tuberous sclerosis complex tumor-suppressor genes (TSC1/TSC2) that cause LAM, a multisystem disease characterized by cystic lung destruction, lymphatic infiltration, and abdominal tumors. Herein, we show that OPG stimulated proliferation of cells cultured from explanted LAM lungs, and selectively induced migration of LAM cells identified by the loss of heterozygosity for TSC2. Consistent with these observations, cells with TSC2 loss of heterozygosity expressed the OPG receptors, receptor activator of NF-κB ligand, syndecan-1, and syndecan-2. LAM lung nodules showed reactivities to antibodies to tumor necrosis factor-related apoptosis-inducing ligand, receptor activator of NF-κB ligand, syndecan-1, and syndecan-2. LAM lung nodules also produced OPG, as shown by expression of OPG mRNA and colocalization of reactivities to anti-OPG and anti-gp100 (HMB45) antibodies in LAM lung nodules. Serum OPG was significantly higher in LAM patients than in normal volunteers. Based on these data, it appears that OPG may have tumor-promoting roles in the pathogenesis of lymphangioleiomyomatosis, perhaps acting as both autocrine and paracrine factors.

Topics: Adult; Cell Movement; Cell Proliferation; Cells, Cultured; Chemokine CCL2; Flow Cytometry; gp100 Melanoma Antigen; Healthy Volunteers; Humans; Loss of Heterozygosity; Lung; Lymphangioleiomyomatosis; Microdissection; Middle Aged; Neoplasm Metastasis; Osteoprotegerin; Receptors, Cell Surface; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

The estrogen receptor (ER)β1 is successively lost during cancer progression, whereas its splice variant, ERβ2, is expressed in advanced prostate cancer. The latter form of cancer often metastasizes to bone, and we wanted to investigate whether the loss of ERβ1 and/or the expression of ERβ2 affect such signaling pathways in prostate cancer. Using PC3 and 22Rv1 prostate cancer cell lines that stably express ERβ1 or ERβ2, we found that the ERβ variants differentially regulate genes known to affect tumor behavior. We found that ERβ1 repressed the expression of the bone metastasis regulator Runx2 in PC3 cells. By contrast, RUNX2 expression was up-regulated at the mRNA level by ERβ2 in PC3 cells, whereas Slug was up-regulated by ERβ2 in both PC3 and 22Rv1 cells. In addition, the expression of Twist1, a factor whose expression strongly correlates with high Gleason grade prostate carcinoma, was increased by ERβ2. In agreement with the increased Twist1 expression, we found increased expression of Dickkopf homolog 1; Dickkopf homolog 1 is a factor that has been shown to increase the RANK ligand/osteoprotegerin ratio and enhance osteoclastogenesis, indicating that the expression of ERβ2 can cause osteolytic cancer. Furthermore, we found that only ERβ1 inhibited proliferation, whereas ERβ2 increased proliferation. The expression of the proliferation markers Cyclin E, c-Myc, and p45(Skp2) was differentially affected by ERβ1 and ERβ2 expression. In addition, nuclear β-catenin protein and its mRNA levels were reduced by ERβ1 expression. In conclusion, we found that ERβ1 inhibited proliferation and factors known to be involved in bone metastasis, whereas ERβ2 increased proliferation and up-regulated factors involved in bone metastasis. Thus, in prostate cancer cells, ERβ2 has oncogenic abilities that are in strong contrast to the tumor-suppressing effects of ERβ1.

Topics: Animals; Bone Neoplasms; Cell Line, Tumor; Cell Proliferation; Core Binding Factor Alpha 1 Subunit; Estrogen Receptor beta; Humans; Intercellular Signaling Peptides and Proteins; Male; Mice; Mice, Nude; Neoplasm Metastasis; Nuclear Proteins; Osteoclasts; Osteoprotegerin; Prostatic Neoplasms; Protein Isoforms; RANK Ligand; RNA, Messenger; Signal Transduction; Snail Family Transcription Factors; Transcription Factors; Transcriptional Activation; Twist-Related Protein 1

This study aimed to identify a novel biomarker or a target of treatment for colorectal cancer (CRC).. The expression profiles of cancer cells in 104 patients with CRC were examined using laser microdissection and oligonucleotide microarray analysis. Overexpression in CRC cells, especially in patients with distant metastases, was a prerequisite to select candidate genes. The mRNA expression of candidate genes was investigated by quantitative reverse transcriptase PCR (RT-PCR) in 77 patients as a validation study. We analyzed the protein expression and localization of the candidate gene by immunohistochemical study and investigated the relationship between protein expression and clinicopathologic features in 274 CRC patients.. Using microarray analysis, we identified 6 candidate genes related to distant metastases in CRC patients. Among these genes, osteoprotegerin (OPG) is known to be associated with aggressiveness in several cancers through inhibition of apoptosis via neutralization of the function of TNF-related apoptosis-inducing ligand. The mRNA expression of OPG in cancer tissues was significantly higher in patients with distant metastases than those without metastases. Overexpression of OPG protein was associated with significantly worse overall survival and relapse-free survival. Moreover, overexpression of the OPG protein was an independent risk factor for CRC recurrence.. Overexpression of OPG may be a predictive biomarker of CRC recurrence and a target for treatment of this disease.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Colorectal Neoplasms; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genetic Predisposition to Disease; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Male; Middle Aged; Neoplasm Metastasis; Neoplasm Recurrence, Local; Oligonucleotide Array Sequence Analysis; Osteoprotegerin; Proportional Hazards Models; Reverse Transcriptase Polymerase Chain Reaction; Young Adult

Receptor activator of NFkB (RANK), its ligand (RANKL) and the decoy receptor of RANKL (osteoprotegerin, OPG) play a pivotal role in bone remodeling by regulating osteoclasts formation and activity. RANKL stimulates migration of RANK-expressing tumor cells in vitro, conversely inhibited by OPG.. We examined mRNA expression levels of RANKL/RANK/OPG in a publicly available microarray dataset of 295 primary breast cancer patients. We next analyzed RANK expression by immunohistochemistry in an independent series of 93 primary breast cancer specimens and investigated a possible association with clinicopathological parameters, bone recurrence and survival.. Microarray analysis showed that lower RANK and high OPG mRNA levels correlate with longer overall survival (P = 0.0078 and 0.0335, respectively) and disease-free survival (P = 0.059 and 0.0402, respectively). Immunohistochemical analysis of RANK showed a positive correlation with the development of bone metastases (P = 0.023) and a shorter skeletal disease-free survival (SDFS, P = 0.037). Specifically, univariate analysis of survival showed that "RANK-negative" and "RANK-positive" patients had a SDFS of 105.7 months (95% CI: 73.9-124.4) and 58.9 months (95% CI: 34.7-68.5), respectively. RANK protein expression was also associated with accelerated bone metastasis formation in a multivariate analysis (P = 0.029).. This is the first demonstration of the role of RANK expression in primary tumors as a predictive marker of bone metastasis occurrence and SDFS in a large population of breast cancer patients.

Topics: Aged; Bone Neoplasms; Breast Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Ligands; Middle Aged; Neoplasm Metastasis; Oligonucleotide Array Sequence Analysis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; RNA, Messenger

This is a retrospective study on 40 breast cancer patients, of which 20 have bone metastases, 10 have visceral metastases, and 10 have no evidence of disease, aimed at evaluating the role of CXCR4 and the RANK/RANKL/OPG system to predict bone metastases. CXCR4 expression, alone or in combination with RANK, identified patients destined to relapse to bone.. The RANK/RANKL/OPG system is active in primary cancers such as breast, prostate, and also in their bone metastases. CXCR4 chemokine receptor is highly expressed in human breast cancer cells and is believed to facilitate the homing of tumor cells to organs such as bone that express high levels of its ligand SDF1. Our study aimed to investigate whether the analysis of these markers with an inexpensive and simple test can help to predict bone metastases in breast cancer patients.. Marker expression was evaluated by immunohistochemical staining in paraffin-embedded tissue sections of primary breast cancers from 40 individuals: 20 patients with bone metastases (BM), 10 with visceral metastases (VM; considered together as the relapsed group), and 10 with no evidence of disease (NED).. RANKL was not detected in tumor cells. OPG- and RANK-positive tumors are found with similar frequency in NED (20%) and in relapsed patients (23% and 17%, respectively). However, in the latter subgroup, only RANK positivity was always associated with bone relapse. The frequency of CXCR4-positive tumors was three-fold higher in relapsed (30%) than in NED (10%) patients and positivity was always linked to bone metastases. Considering NED and VM patients together versus BM patients, we observed that CXCR4 expression, alone (P = .008) or in combination with RANK (P < .001), identified patients destined to relapse to bone.. Our results provide the first clinical evidence to support a pivotal role of combined CXCR4 and RANK expression in predicting bone relapse.

Topics: Aged; Aged, 80 and over; Biomarkers, Tumor; Bone Neoplasms; Breast Neoplasms; Case-Control Studies; Female; Humans; Middle Aged; Neoplasm Metastasis; Osteoprotegerin; Predictive Value of Tests; RANK Ligand; Receptors, CXCR4; Retrospective Studies

To find the optimal proportion of Composite Fructus Psoralea and Fructus Cnidii (CFPC) for inhibiting the bone metastasis of breast cancer by way of exploring its acting mechanism viewing from OPG/RANKL/RANK system.. The human bone metastasis of breast cancer model was established by injecting tumor cells of MDA-MB-231BO cell line into the left cardiac ventricle of nude mice. The modeled mice were randomly divided into seven groups: the blank group administered with normal saline by gastrogavage, the positive control group with zoledronic acid via peritoneal injection, and the 5 tested group with CFPC in different proportions of Fructus Psoralea and Fructus Cnidii, i.e., (A, 4:0; B, 3:1; C, 1:1; D, 1:3, and E 0:4), given by gastric infusion. The treatment started from 1 week after modeling and lasted for six weeks. By the end of the experiment, the metastatic foci in bone were imaged by radionuclide tracing method and X-ray photograph, and separated for detecting gene and protein expressions of osteoprotegerin (OPG), receptor activator of nuclear factor-kappaB ligand (RANKL), interleukin-8 (IL-8), parathyroid hormone-related protein (PTHrP), macrophage colony stimulating factor (MCSF) by Real-time PCR and Western blot respectively.. Inhibition of bone metastasis gene was displayed to some extent in all the tested groups treated with CFPC, showing an increased level of OPG mRNA expression (It was 60.343 +/- 6.274 in the tested group C), and decreased mRNA expressions of IL-8, PTHrP, MCSF, RANKL (218.010 +/- 12.802, 232.399 +/- 14.354, 319.831 +/- 5.322, and 195.701 +/- 4. 862, respectively in the tested group C). The optimal effect was shown in the tested group C, showing significant difference to that in the blank group (P < 0.01). Meanwhile, the OPG in the bone metastatic foci could be up-regulated and protein expressions of RANKL/IL-8/PTHrP/MCSF down-regulated in all the tested groups. The optimal effect was shown in the tested group C, with significant difference from those of the normal saline group.. CFPC could inhibit the bone metastasis of breast cancer through activating OPG/RANKL/RANK pathway. Among different proportions of Fructus Psoralea and Fructus Cnidii, 1:1 was the best one.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Coumarins; Female; Ficusin; Interleukin-8; Macrophage Colony-Stimulating Factor; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Osteoprotegerin; Parathyroid Hormone-Related Protein; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B

Bone metastases cause severe skeletal complications and are associated with osteoclast-mediated bone destruction. RANKL is essential for osteoclast formation, function, and survival, and is the primary effector of tumor-induced osteoclastogenesis and osteolysis. RANKL inhibition by its soluble decoy receptor osteoprotegerin (OPG) prevents tumor-induced osteolysis and decreases skeletal tumor burden. Because osteoclast-mediated bone resorption releases growth factors from the bone matrix, the host bone microenvironment induces a vicious cycle of bone destruction and tumor proliferation and survival. A prediction of this vicious cycle hypothesis is that targeting the host bone microenvironment by osteoclast inhibition would reduce tumor growth and survival and may enhance the anti-tumor effects of targeted therapies. The epidermal growth factor receptor (EGFR) pathway regulates critical processes such as cell growth and survival, and anti-EGFR therapies can cause tumor cell arrest and apoptosis. We evaluated whether reduction of osteolysis by RANKL inhibition could enhance the anti-tumor effects of an anti-EGFR antibody (panitumumab) in a novel murine model of human A431 epidermoid carcinoma bone metastasis. Skeletal tumor progression was assessed longitudinally by bioluminescence imaging. RANKL inhibition by OPG-Fc treatment resulted in a reduction in tumor progression in bony sites. OPG-Fc treatment also caused a dose-dependent reduction in tumor-induced osteolysis, supporting the essential role of RANKL in this process. In combination, RANKL inhibition increased the anti-tumor efficacy of an anti-EGFR antibody, and completely blocked tumor-induced bone breakdown, demonstrating that addition of the indirect anti-tumor effect of RANKL inhibition increases the anti-tumor efficacy of panitumumab, a targeted anti-EGFR antibody.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; Bone Neoplasms; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; ErbB Receptors; Humans; Mice; Mice, Nude; Neoplasm Metastasis; Osteoprotegerin; Panitumumab; Random Allocation; RANK Ligand

cAMP-response-element-binding protein (CREB) signaling has been reported to be associated with cancer development and poor clinical outcome in various types of cancer. However, it remains to be elucidated whether CREB is involved in breast cancer development and osteotropism. Here, we found that metastatic MDA-MB-231 breast cancer cells exhibited higher CREB expression than did non-metastatic MCF-7 cells and that CREB expression was further increased by several soluble factors linked to cancer progression, such as IL-1, IGF-1, and TGF-beta. Using wild-type CREB and a dominant-negative form (K-CREB), we found that CREB signaling positively regulated the proliferation, migration, and invasion of MDA-MB-231 cells. In addition, K-CREB prevented MDA-MB-231 cell-induced osteolytic lesions in a mouse model of cancer metastasis. Furthermore, CREB signaling in cancer cells regulated the gene expression of PTHrP, MMPs, and OPG, which are closely involved in cancer metastasis and bone destruction. These results indicate that breast cancer cells acquire CREB overexpression during their development and that this CREB upregulation plays an important role in multiple steps of breast cancer bone metastasis.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Cyclic AMP Response Element-Binding Protein; Female; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor I; Interleukin-17; Matrix Metalloproteinases; Mice; Mice, Nude; Neoplasm Metastasis; Osteolysis; Osteoprotegerin; Parathyroid Hormone-Related Protein; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

During breast cancer metastasis to bone, tumor cells home to bone marrow, likely targeting the stem cell niche, and stimulate osteoclasts, which mediate osteolysis required for tumor expansion. Although osteoblasts contribute to the regulation of the hematopoietic stem cell niche and control osteoclastogenesis through production of proresorptive cytokine RANKL (receptor activator of NF-κB ligand), their role in cancer metastases to bone is not fully understood. C57BL/6J mouse bone marrow cells were treated for 3-12 days with ascorbic acid (50 μg/ml) in the presence or absence of 10% medium conditioned by breast carcinoma cells MDA-MB-231, 4T1, or MCF7. Treatment with cancer-derived factors resulted in a sustained 40-60% decrease in osteoblast differentiation markers, compared with treatment with ascorbic acid alone, and induced an osteoclastogenic change in the RANKL/osteoprotegerin ratio. Importantly, exposure of bone cells to breast cancer-derived factors stimulated the subsequent attachment of cancer cells to immature osteoblasts. Inhibition of γ-secretase using pharmacological inhibitors DAPT and Compound E completely reversed cancer-induced osteoclastogenesis as well as cancer-induced enhancement of cancer cell attachment, identifying γ-secretase activity as a key mediator of these effects. Thus, we have uncovered osteoblasts as critical intermediary of premetastatic signaling by breast cancer cells and pinpointed γ-secretase as a robust target for developing therapeutics potentially capable of reducing both homing and progression of cancer metastases to bone.

Topics: Amyloid Precursor Protein Secretases; Animals; Antigens, Differentiation; Antioxidants; Ascorbic Acid; Benzodiazepinones; Bone Marrow Cells; Bone Neoplasms; Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Culture Media, Conditioned; Female; Humans; Mammary Neoplasms, Animal; Mice; Neoplasm Metastasis; Osteoblasts; Osteoclasts; Osteoprotegerin; RANK Ligand; Signal Transduction

Osteoprotegerin (OPG) inhibits osteoclast activation and reduces osteolysis in bone tumors. We hypothesized that tumor-tropic neural progenitor cells (NPCs) engineered to express OPG would reduce neuroblastoma disease burden in the bone.. Stable expression of green fluorescent protein (NPC-GFP) and OPG (NPC-OPG) was established in human NPCs by lentivirus-mediated transduction. Bone disease was established by intrafemoral injection of luciferase-expressing human neuroblastoma (CHLA-255) cells into 20 SCID mice. Three weeks later, mice began receiving intravenous injection of 2 x 10(6) NPC-OPG or NPC-GFP (control) every 10 days x 3 doses. Disease was monitored with quantitative bioluminescence imaging and x-ray images, which were evaluated on a scale of 0 to 4. These studies were approved by the Institutional Animal Care and Use Committee.. Osteoprotegerin treatment in vitro produced no direct toxicity to tumor cells. Coculture of tumor cells with bone marrow significantly increased activation of bone marrow-derived osteoclasts as assessed by tartrate-resistant acid phosphatase staining (156 +/- 10.8 osteoclasts per well) compared to bone marrow culture alone (91.67 +/- 4.7, P = .005). This increase was abrogated by adding OPG-containing media (68.3 +/- 2.8, P = .001). NPC-OPG slowed tumor progression (108-fold increase from pretreatment) compared to mice treated with NPC-GFP (538-fold), as judged by bioluminescence imaging. X-rays subjectively demonstrated less bone disease in NPC-OPG-treated mice (2.27 +/- 0.25) compared to NPC-GFP-treated mice (3.25 +/- 0.22, P = .04).. Neural progenitor cell-mediated delivery of OPG slowed disease progression in a preclinical model of neuroblastoma bone metastasis. The decrease in bone disease was not from direct tumor cell toxicity but likely occurred indirectly through inhibition of osteoclast-directed bone resorption. Thus, targeted delivery of OPG by NPCs may be effective in the treatment of neuroblastoma bone metastasis.

Topics: Animals; Bone Neoplasms; Cell Line, Tumor; Disease Progression; Green Fluorescent Proteins; Humans; In Vitro Techniques; Mice; Mice, SCID; Neoplasm Metastasis; Neuroblastoma; Osteoprotegerin; Statistics, Nonparametric; Stem Cells; Transduction, Genetic

Receptor activator of NF-kappaB ligand (RANKL) and its receptor, receptor activator of NF-kappaB (RANK), play a key role in osteoclastogenesis, and osteoprotegerin (OPG) acts as a decoy receptor for RANKL. We investigated the role of the RANKL-RANK-OPG system in renal cell carcinomas (RCCs), which frequently metastasize to bones. Real-time quantitative PCR revealed that RANKL mRNA expression was higher in clear cell RCCs than in papillary and chromophobe RCCs. Similarly, RANKL protein expression level in clear cell RCCs was higher than that in papillary and chromophobe RCCs, showing positive correlations with the primary tumour stage and distant metastasis. There was no significant association between the expression level of RANK, OPG and histological subtypes of RCC. RANKL and RANK expression was observed in metastatic RCCs in the bone and other organs, suggesting that they play a role in metastasis to the bone and other organs. Recombinant RANKL protein stimulated migration of a clear cell RCC cell line, Caki-1, in vitro, and this enhanced migration was inhibited by the administration of recombinant OPG protein. Furthermore, multivariate Cox analysis revealed that elevated RANKL and RANK expression with low-OPG expression was a significant and independent predictor of recurrence, bone metastasis and a poor prognosis. These data suggest that the RANKL-RANK-OPG system is involved not only in the bone metastasis of RCCs but also in metastasis to other organs through the stimulation of cancer cell migration.

Topics: Bone Neoplasms; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Movement; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Kidney Neoplasms; Male; Middle Aged; Neoplasm Metastasis; Osteoprotegerin; Prognosis; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Statistics, Nonparametric; Survival Analysis; Up-Regulation

Current evidence indicates that an osteoblast lesion in prostate cancer is preceded by osteolysis. Thus, prevention of osteolysis would reduce complications of bone metastasis. Bone marrow-derived mesenchymal stem cells have the ability to differentiate into osteoblast and produce osteoprotegerin, a decoy receptor for the receptor activator for nuclear factor kappaB ligand, naturally. The present study examined the potential of unmodified mesenchymal stem cells to prevent osteolytic bone lesions in a preclinical mouse model of prostate cancer.. The human prostate cancer cell line PC3 was implanted in tibiae of severe combined immunodeficient mice. After establishment of the tumor, either unmodified or genetically engineered mesenchymal stem cells overexpressing osteoprotegerin was injected at the site of tumor growth. The effects of therapy were monitored by bioluminescence imaging, micro-computed tomography, immunohistochemistry, and histomorphometry.. Data indicated significant (P < 0.001) inhibition of tumor growth and restoration of bone in mice treated with unmodified and modified mesenchymal stem cells. Detailed analysis suggested that the donor mesenchymal stem cell inhibited tumor progression by producing woven bone around the growing tumor cells in the tibiae and by preventing osteoclastogenesis.. Overcoming the limitation of the number of mesenchymal stem cells available in the bone can provide significant amelioration for osteolytic damage without further modification.

Topics: Animals; Bone Marrow Cells; Cell Line, Tumor; Cell- and Tissue-Based Therapy; Humans; Immunohistochemistry; Male; Mesenchymal Stem Cells; Mice; Mice, SCID; Neoplasm Metastasis; Osteoclasts; Osteoprotegerin; Prostatic Neoplasms; Tibia; X-Ray Microtomography

Resistance to apoptosis is a hallmark of cancer and correlates with aggressiveness of tumors and poor prognosis. The Wnt/beta-catenin pathway plays a pivotal role in the genesis of colorectal cancer by mechanisms not fully elucidated yet. Previous studies have linked regulation of osteoprotegerin (OPG) in bone to Wnt/beta-catenin signaling. As OPG also serves as a decoy receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), we hypothesized that OPG might play a role in mediating resistance to apoptosis in colorectal cancer cells.. Expression analysis and functional studies in human colorectal cancer cell lines and determination of expression in primary tumors and sera from patients with colorectal cancer.. We found production of OPG in colorectal cancer cells to be regulated by beta-catenin/Tcf-4. Addition of exogenous OPG to colorectal cancer cells caused resistance to TRAIL. Similarly, accumulation of OPG in medium of cultivated cells caused resistance to TRAIL, and this could be reverted by removal of OPG. Furthermore, OPG levels were significantly increased in serum of patients with advanced disease.. We conclude that the Wnt/beta-catenin pathway contributes to carcinogenesis and cancer cell survival by driving expression of OPG. Expression of the survival factor OPG might provide colorectal cancer cells with an essential growth advantage and contribute to cell invasion and metastasis. Inhibition of OPG expression might offer a new therapeutic approach for the treatment of patients with colorectal tumors overexpressing OPG and make these tumors sensitive to TRAIL-induced apoptosis.

Topics: Apoptosis; beta Catenin; Cell Line, Tumor; Colonic Neoplasms; Colorectal Neoplasms; Disease Progression; Drug Resistance, Neoplasm; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Osteoprotegerin; RNA, Small Interfering; TNF-Related Apoptosis-Inducing Ligand

To clarify the mechanisms of bone destruction associated with bone metastases, we studied an animal model in which inoculation of MDA-MB-231 human breast cancer cells into the left cardiac ventricle of female nude mice causes osteolytic lesions in bone using morphological techniques. On the bone surfaces facing the metastatic tumor cells, there existed many tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts. TRAP-positive mononuclear osteoclast precursor cells were also observed in the tumor nests. Immunohistochemical studies showed that the cancer cells produced parathyroid hormone-related protein (PTHrP) but not receptor activator of NF-kappaB ligand (RANKL). Histochemical and immunohistochemical examinations demonstrated that alkaline phosphatase and RANKL-positive stromal cells were frequently adjacent to TRAP-positive osteoclast-like cells. Immunoelectron microscopic observation revealed that osteoclast-like cells were in contact with RANKL-positive stromal cells. MDA-MB-231 cells and osteoclast-like cells in the tumor nests showed CD44-positive reactivity on their plasma membranes. Hyaluronan (HA) and osteopontin (OPN), the ligands for CD44, were occasionally colocalized with CD44. These results suggest that tumor-producing osteoclastogenic factors, including PTHrP, upregulate RANKL expression in bone marrow stromal cells, which in turn stimulates the differentiation and activation of osteoclasts, leading to the progression of bone destruction in the bone metastases of MDA-MB-231 cells. Because the interactions between CD44 and its ligands, HA and OPN, have been shown to upregulate osteoclast differentiation and function, in addition to the cell-cell interactions mediated by RANK and RANKL, the cell-matrix interactions mediated by these molecules may also contribute to the progression of osteoclastic bone destruction.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Cell Communication; Cell Line, Tumor; Cytokines; Disease Models, Animal; Extracellular Matrix; Female; Humans; Hyaluronan Receptors; Hyaluronic Acid; Mice; Mice, Nude; Neoplasm Metastasis; Osteoclasts; Osteoprotegerin; Parathyroid Hormone-Related Protein; RANK Ligand

Enhanced production of receptor activator of nuclear factor-kappaB ligand (RANKL) and its binding to RANK on the osteoclasts have been associated with osteolysis in breast cancer bone metastasis. Osteoprotegerin (OPG) is a decoy receptor that prevents RANKL-RANK interaction. This study determined the effects of sustained expression of OPG using a recombinant adeno-associated viral (rAAV) vector in mouse model of osteolytic breast cancer. Bone metastasis was established by intracardiac injection of the human breast cancer cell line MDA-MB-435. Following this, mice were administered a one-time intramuscular injection of rAAV encoding either OPG.Fc (OPG) or green fluorescent protein (GFP). Mice were killed 1 month later and the effects of therapy on tumor growth and bone remodeling were evaluated. Bioluminescence imaging showed significant reduction of tumor growth in bone of OPG.Fc-treated mice. Micro-computed tomography (microCT) analysis and histomorphometry of the tibia indicated significant protection of trabecular and cortical bones after OPG.Fc therapy. Despite the prevention of bone loss and tumor growth in bone, OPG.Fc therapy failed to provide long-term survival. OPG.Fc-treated mice developed more bone than age-matched normal mice, indicating a requirement for regulated transgene expression. Results of this study indicate the potential of rAAV-OPG therapy for reducing morbidity and mortality in breast cancer patients with osteolytic bone damage.

Topics: Animals; Bone and Bones; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Dependovirus; Female; Genetic Therapy; Humans; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Osteoprotegerin; RANK Ligand; Tomography, X-Ray Computed

The RANK/RANKL signalling mechanism is the final common pathway of osteoclast formation and activity. Inhibitors of RANK ligand (RANKL) that bind to RANK (for 'receptor activator of NF-kappaB'), such as osteoprotegerin (OPG), neutralizing antibodies against RANKL and soluble RANK antagonists, are well described inhibitors of bone metastasis in preclinical and clinical models, presumably because of their effects on osteoclasts. Jones et al. show that OPG inhibits bone metastasis after intracardiac injection of B16F10 murine melanoma cells, but claim that bone metastases are entirely independent of osteoclast formation and bone resorption: rather, they are caused by an effect on cell migration through RANK. However, we question whether these surprising conclusions are rigorously supported by their data.

Topics: Animals; Bone Neoplasms; Cell Line, Tumor; Cell Movement; Humans; Melanoma; Mice; Neoplasm Metastasis; Osteoclasts; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Reproducibility of Results

Topics: Animals; Bone Neoplasms; Carrier Proteins; Glycoproteins; Humans; Membrane Glycoproteins; Mice; Neoplasm Metastasis; Neoplasms; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, CXCR4; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Late-stage prostate cancer patients are refractory to hormone therapy and exhibit a high propensity to develop skeletal metastasis. In this regard, the role of a novel cytokine system belonging to the tumor necrosis factor (TNF) family that is critical for osteoclastic osteolysis and that consists of receptor activator of NF-kappaB ligand (RANKL), its receptor (RANK), and decoy receptor osteoprotegerin (OPG) is of potential interest.. Reverse-transcriptase polymerase chain reaction (RT-PCR) and immunohistochemical analysis was used to examine the expression of RANKL, RANK, and OPG in human prostate cancer cell lines and in 89 archival samples of primary and metastatic (lymph nodes, skeleton) prostate cancer patients. Expression of these proteins was correlated with clinicopathogic parameters of the prostate cancer.. Expression of RANKL/RANK/OPG was low in normal but markedly higher in prostate cancer cell lines. Analysis of surgical biopsy specimens showed the expression of RANKL (31%), RANK (38%), and OPG (19%) in primary carcinoma. The expression frequency was significantly higher (RANKL [44%], RANK [49%], and OPG [73%]) in metastatic prostate cancer. OPG (83%) production was more common in skeletal as compared with lymph node metastases (46%), whereas the expression of RANKL expression was less discordant in bone (47%) and lymph node metastases (36%). The increased expression of RANKL/RANK/OPG observed correlated with Gleason score, TNM stage, androgen status, and serum prostate-specific antigen (PSA) levels in the prostate cancer patients.. Expression of RANKL/RANK/OPG correlates with more aggressive, advanced, metastatic prostate carcinoma to suggest their role as diagnostic, prognostic, and therapeutic targets for prostate cancer.

Topics: Aged; Biomarkers, Tumor; Carcinoma; Carrier Proteins; Glycoproteins; Humans; Male; Membrane Glycoproteins; Middle Aged; Neoplasm Metastasis; Neoplasm Staging; Osteoprotegerin; Prognosis; Prostate-Specific Antigen; Prostatic Neoplasms; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Tumor Cells, Cultured; Up-Regulation

During normal bone remodeling, the receptor activator of nuclear factor-kappaB (RANK) interacts with its ligand RANKL, which is present on pre-osteoclasts, resulting in bone resorption and initiation of new bone formation. When breast cancer metastasizes to bone, normal bone remodeling is disturbed by invasion of tumor cells, resulting in osteolytic lesions. We have studied the expression of both RANK and RANKL in 10 nonneoplastic breast samples, 58 infiltrating ductal carcinoma (IDC), and 43 breast cancer bony metastases (BTM). RANK seemed to be present in all samples tested. However, whereas RANKL expression was observed in 90% of nonneoplastic breast, RANKL expression was only observed in 62% of nonmetastatic IDC, 31% of metastatic IDC, and 2% of osteolytic BTM lesions. This decreased or absent expression of RANKL in the tumor cells may allow RANK, which is normally expressed as a receptor on the cell surface, to target RANKL present on the cell surface of normal osteoblasts and stromal cells of the bone. Stimulation of the normal osteoblasts and stromal cells by the tumor cells may then lead to secondary osteoclastogenesis, resulting in the osteolytic phenotype common to breast metastases.

Topics: Bone and Bones; Bone Remodeling; Breast; Breast Neoplasms; Carcinoma, Ductal, Breast; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Immunohistochemistry; Neoplasm Metastasis; Osteoblasts; Osteoclasts; Osteoprotegerin; Phenotype; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Time Factors

Zoledronic acid (ZA) has been shown to inhibit prostate tumor growth in vitro and have beneficial effects in patients with advanced prostate cancer (CaP). The aim of this study was to determine whether ZA exhibits direct anti-tumor effects on CaP cells in vivo. To distinguish the effects of inhibition of osteolysis and direct anti-tumor activity of ZA in vivo, we compared the results of treatment with ZA and osteoprotegerin (Fc-OPG), which inhibits osteolysis, but without significant direct anti-tumor effects. In vitro Fc-OPG had no significant effects on C4-2 proliferation, whereas ZA decreased proliferation. However, both agents decreased tumor growth in bone. Moreover, both increased bone volume and prevented the overall decreases in BMD associated with growth of C4-2 cells in bone. Our study provides novel and significant observations that the in vivo effects of ZA are consistent with indirect effects mediated by osteoclasts.

Topics: Animals; Antibodies; Antineoplastic Agents; Apoptosis; Bone Density; Bone Density Conservation Agents; Bone Neoplasms; Cell Proliferation; Densitometry; Diphosphonates; Glycoproteins; Humans; Imidazoles; Immunoglobulin Fc Fragments; In Vitro Techniques; Male; Mice; Mice, SCID; Neoplasm Metastasis; Osteoclasts; Osteolysis; Osteoprotegerin; Prostatic Neoplasms; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Tibia; Time Factors; Zoledronic Acid

Prostate cancer specifically metastasizes to bone where it leads to bone formation. We previously reported that coculturing MDA PCa 2b prostate cancer cells with primary mouse osteoblasts (PMOs) induced PMO proliferation and differentiation. An osteoblastic reaction was also observed in vivo after injection of MDA PCa 2b cells into the bones of severe combined immunodeficient disease mice. The aim of this study was to identify the sequence of events that leads to these osteoblastic lesions in vivo and, using this in vitro model, to define the contributions of various genes and cellular pathways in the pathophysiology of osteoblastic bone metastases of prostate cancer.. We show histological evidence of de novo bone formation as early as 2 weeks after injection of MDA PCa 2b cells in the bone of severe combined immunodeficient disease mice. In vitro, we show that PMOs induce MDA PCa 2b proliferation, suggesting a synergistic paracrine loop between these cells and PMOs. Endothelin (ET)-1, which is a mitogen for several cell types, is produced by all prostate cancer cell lines tested, and Atrasentan, an antagonist of ET-1 receptor A, partially reversed PMO proliferation induced by MDA PCa 2b cells. ET-1 is known to be comitogenic with a number of growth factors, including insulin-like growth factor (IGF)-I. In this study, we report that IGF-binding protein (IGFBP)-3 transcripts (that regulate levels of free IGF) are down-regulated in prostate cancer cells cocultured with PMO, whereas prostate-specific antigen (a protease known to cleave IGFBP-3) is detected in the 150-400 ng/ml range. Accordingly, IGFBP-3 has antiproliferative effects in PMOs, which were attenuated in our in vitro system. Taken together, our studies also implicate the IGF axis to play a role in this model of bone metastases. Secondly, the transcript level of mouse double minute 2 (a protein that regulate p53) was increased in prostate cancer cells grown with PMOs. The p53-dependent and -independent oncogenic activities of mouse double minute 2 suggest that osteoblasts induce a survival advantage in prostate cancer cells. Lastly, we show that expression of osteoprotegerin is decreased and of receptor activator of nuclear factor-kappaB ligand is increased in PMOs cultured in the presence of MDA PCa 2b cells, two events associated with osteoclast activation and bone resorption.. Our results provide evidence that multiple and distinct molecular events affecting both bone formation and bone resorption concur to the increase bone mass in prostate cancer bone metastases. These data also provide a rationale for developing therapeutic strategies designed to target these molecular changes.

Topics: Alkaline Phosphatase; Animals; Blotting, Northern; Carrier Proteins; Cell Differentiation; Cell Division; Cell Line, Tumor; Cell Survival; Coculture Techniques; Culture Media, Conditioned; DNA; DNA, Complementary; Down-Regulation; Glycoproteins; Humans; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor I; Male; Membrane Glycoproteins; Mice; Mice, SCID; Models, Biological; Neoplasm Metastasis; Oligonucleotide Array Sequence Analysis; Osteoblasts; Osteoprotegerin; Phenotype; Prostatic Neoplasms; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; RNA; RNA, Messenger

Bone destruction is primarily mediated by osteoclastic bone resorption, and cancer cells stimulate the formation and activation of osteoclasts next to metastatic foci. Accumulating evidences indicate that receptor activator of NF-kB ligand (RANKL) is the ultimate extracellular mediator that stimulates osteoclast differentiation into mature osteoclasts. In contrast, osteoprotegerin (OPG) inhibits osteoclast development. In order to elucidate a mechanism for cancer-induced osteoclastogenesis, cells from a human breast cancer line, MDA-MB-231, were directly co-cultured with ST2, MC3T3-E1, or with primary mouse calvarial cells. Osteoclast-like cells and tartarate resistant acid phosphatase (TRAP) activities were then quantitated. We examined these cell lines and samples from breast cancer by RT-PCR for the expressions of OPG and RANKL mRNA. Compared to controls, co-culture of MDA-MB-231 cells with stromal or osteoblastic cells induced an increase in number of osteoclasts and TRAP activities. MDA-MB-231 cells alone or breast cancer samples did not express RANKL mRNA. However, co-culture of these cancer cells with stromal or osteoblastic cells induced RANKL mRNA expression and decreased OPG mRNA expression. These experiments demonstrate that direct interactions between breast cancer and stromal or osteoblastic cells induce osteoclastogenesis in vitro through modulating RANKL expression.

Topics: 3T3 Cells; Acid Phosphatase; Animals; Bone Neoplasms; Breast Neoplasms; Carrier Proteins; Cell Differentiation; Cell Line, Tumor; Cells, Cultured; Coculture Techniques; Culture Media, Conditioned; Glycoproteins; Humans; Isoenzymes; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Osteoblasts; Osteoclasts; Osteoprotegerin; Protein Binding; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tartrate-Resistant Acid Phosphatase; Time Factors

Prostate cancer (CaP) forms osteoblastic skeletal metastases with an underlying osteoclastic component. However, the importance of osteoclastogenesis in the development of CaP skeletal lesions is unknown. In the present study, we demonstrate that CaP cells directly induce osteoclastogenesis from osteoclast precursors in the absence of underlying stroma in vitro. CaP cells produced a soluble form of receptor activator of NF-kappaB ligand (RANKL), which accounted for the CaP-mediated osteoclastogenesis. To evaluate for the importance of osteoclastogenesis on CaP tumor development in vivo, CaP cells were injected both intratibially and subcutaneously in the same mice, followed by administration of the decoy receptor for RANKL, osteoprotegerin (OPG). OPG completely prevented the establishment of mixed osteolytic/osteoblastic tibial tumors, as were observed in vehicle-treated animals, but it had no effect on subcutaneous tumor growth. Consistent with the role of osteoclasts in tumor development, osteoclast numbers were elevated at the bone/tumor interface in the vehicle-treated mice compared with the normal values in the OPG-treated mice. Furthermore, OPG had no effect on CaP cell viability, proliferation, or basal apoptotic rate in vitro. These results emphasize the important role that osteoclast activity plays in the establishment of CaP skeletal metastases, including those with an osteoblastic component.

Topics: Animals; Bone Neoplasms; Bone Resorption; Carrier Proteins; Glycoproteins; Humans; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, SCID; Neoplasm Metastasis; Osteoprotegerin; Prostatic Neoplasms; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Tibia