osteoprotegerin and Breast-Neoplasms

RANKL/RANK/OPG system consists of three essential signaling molecules: i) the receptor activator of nuclear factor (NF)-kB-ligand (RANKL), ii) the receptor activator of NF-kB (RANK), and iii) the soluble decoy receptor osteoprotegerin (OPG). Although this system is critical for the regulation of osteoclast differentiation/activation and calcium release from the skeleton, different studies have elucidated its specific role in mammary gland physiology and hormone-driven epithelial proliferation during pregnancy. Of note, several data suggest that progesterone induces mammary RANKL expression in mice and humans. In turn, RANKL controls cell proliferation in breast epithelium under physiological conditions typically associated with higher serum progesterone levels, such as luteal phase of the menstrual cycle and pregnancy. Hence, RANKL/RANK system can be regarded as a major downstream mediator of progesterone-driven mammary epithelial cells proliferation, potentially contributing to breast cancer initiation and progression. Expression of RANKL, RANK, and OPG has been detected in breast cancer cell lines and in human primary breast cancers. To date, dysregulation of RANKL/RANK/OPG system at the skeletal level has been widely documented in the context of metastatic bone disease. In fact, RANKL inhibition through the RANKL-blocking human monoclonal antibody denosumab represents a well-established therapeutic option to prevent skeletal-related events in metastatic bone disease and adjuvant therapy-induced bone loss in breast cancer. On the other hand, the exact role of OPG in breast tumorigenesis is still unclear. This review focuses on molecular mechanisms linking RANKL/RANK/OPG system to mammary tumorigenesis, highlighting pre-clinical and clinical evidence for the potential efficacy of RANKL inhibition as a prevention strategy and adjuvant therapy in breast cancer settings.

Topics: Animals; Bone Neoplasms; Bone Remodeling; Breast Neoplasms; Female; Humans; Mammary Neoplasms, Experimental; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B

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

BRCA1 mutation carriers face a high lifetime risk of breast cancer, estimated at 60 % compared to 10 % in the general population. BRCA1 breast cancers typically have an aggressive course (i.e., high-grade, triple-negative) and are associated with a poor prognosis. At present, primary prevention is limited to prophylactic removal of the unaffected breasts. Effective chemopreventive strategies are not yet available. Emerging evidence suggests that BRCA1 mutation carriers have high circulating levels of progesterone which may play a role in their susceptibility to breast cancer. Recently, the RANK/RANKL system was found to be dysregulated in women with a BRCA1 mutation. Mutation carriers had significantly lower endogenous levels of osteoprotegerin (OPG) than women without a BRCA1 mutation. OPG is an endogenous decoy receptor for RANKL and inhibits RANKL-mediated signaling. RANKL binds to RANK on mammary epithelial cells and stimulates their proliferation and maturation. Low OPG levels may contribute to mammary tumorigenesis through increased proliferation and may explain in part the increased breast cancer risk in BRCA1 mutation carriers. Denosumab is an anti-RANKL monoclonal antibody which is approved to treat osteoporosis and to prevent skeletal damage caused by bone metastases. The emerging role of aberrant RANK-signaling in BRCA1 tumorigenesis suggests that targeting of RANKL may prevent breast cancer among women with germline BRCA1 mutations. Clinical investigations of denosumab are warranted and may lead to a novel chemopreventive approach for breast cancer for high-risk women.

Topics: Biomarkers; BRCA1 Protein; Breast Neoplasms; Cell Transformation, Neoplastic; Chemoprevention; Disease Management; Female; Heterozygote; Humans; Mutation; Osteoprotegerin; Progesterone; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Signal Transduction

Treatment with bisphosphonates and denosumab is the standard of care in bone metastatic disease. In addition, the adjuvant therapy of denosumab or bisphosphonates is very effective to prevent loss of bone mineral density, for example in osteoporosis. However, it is still unclear if this therapy has an influence on preventing cancer.. Since the identification of novel genes in the 1980s, it took about 30 years until denosumab, as a fully human mAb against receptor activator of nuclear factor (NF)-κB ligand (RANKL), could be introduced to clinical practice. The discovery of the receptor activator of NF-κB/RANKL/osteoprotegerin pathway in the 1990s is an example of how modern databases of genes were utilized to discover new pathways relevant to a variety of diseases. The essential role of this pathway for the function, differentiation and survival of osteoclasts, and the influence on the bone microenvironment helped to understand the vicious circle of bone resorption and destruction in many skeletal diseases.. In the following review, we discuss the important role of rational targeting concerning receptor activator of NF-κB/RANKL/osteoprotegerin and the bisphosphonate therapy and provide an update for the related treatment of patients suffering from breast cancer and further implications for clinical practice and research using denosumab as a potential chemoprevention in BRCA1-related breast cancer.

Topics: Bone and Bones; Bone Density Conservation Agents; Bone Neoplasms; Breast Neoplasms; Chemoprevention; Denosumab; Diphosphonates; Female; Genes, BRCA1; Humans; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Risk

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

Osteoprotegerin (OPG) is a secreted protein and member of the Tumor Necrosis Factor (TNF) Receptor superfamily. OPG has been well characterized as a regulator of bone metabolism which acts by blocking osteoclast maturation and preventing bone breakdown. Given this role, early studies on OPG in breast cancer focused on the administration of OPG in order to prevent the osteolysis observed with bone metastases. However OPG is also produced by the breast tumor cells themselves. Research focusing on OPG produced by breast tumor cells has revealed actions of OPG which promote tumor progression. In vitro studies into the role of OPG produced by breast tumor cells have demonstrated that OPG can block TNF-related apoptosis inducing ligand (TRAIL)-mediated apoptosis. Furthermore, in vivo studies show that OPG expression by breast tumors can promote tumor growth and metastasis. In addition it has been shown that OPG stimulates endothelial cell survival and tube formation thus it may indirectly promote breast tumor progression through impacting angiogenesis. This article will present a summary of the data concerning the tumor-promoting effects of OPG in breast cancer.

Topics: Animals; Bone Neoplasms; Bone Remodeling; Breast Neoplasms; Endothelial Cells; Female; Humans; Osteoprotegerin; TNF-Related Apoptosis-Inducing Ligand

RANK and its ligand RANKL are key molecules in bone metabolism and are critically involved in pathologic bone disorders. Deregulation of the RANK/RANKL system is for example a main reason for the development of postmenopausal osteoporosis, which affects millions of women worldwide. Another essential function of RANK and RANKL is the development of a functional lactating mammary gland during pregnancy. Sex hormones, in particular progesterone, induce RANKL expression resulting in proliferation of mammary epithelial cells. Moreover, RANK and RANKL have been shown to regulate mammary epithelial stem cells. RANK and RANKL were also identified as critical mechanism in the development of hormone-induced breast cancer and metastatic spread to bone. In this review, we will focus on the various RANK/RANKL functions ranging from bone physiology, immune regulation, and initiation of breast cancer.

Topics: Animals; Autoimmune Diseases; Bone and Bones; Bone Diseases; Bone Neoplasms; Breast Neoplasms; Cell Transformation, Neoplastic; Female; Humans; Mice; NF-kappa B; Osteoporosis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B

The RANK/RANKL/OPG pathway is well known for bone destruction in skeletal metastases but has also been implicated in osteoclast-independent roles in tumorigenesis and de novo metastasis. Experimental data suggest contribution of progesterone to tumorigenesis may be mediated by RANKL. Importantly, modulation of this pathway became possible through the availability of denosumab, an artificial counterpart of OPG, but significant gaps remain in the translation of preclinical findings on the pathway. We analyzed gene expression of RANK, RANKL and OPG from 40 Affymetrix datasets encompassing 4467 primary breast cancers and focused on ER positive disease. We did not observe a significant prognostic value of RANK and RANKL mRNA expression. In contrast, OPG was associated with a better prognosis among 1941 ER positive cancers (HR 0.64, 95% CI 0.53-0.77; P < 0.0001) using a cutoff from its highly bimodal expression. We detected considerable heterogeneity regarding the prognostic value of OPG between different datasets. This heterogeneity could neither be attributed to technical reasons nor to differences in standard clinical parameters or treatments of the cohorts. Interestingly, the prognostic value of the progesterone receptor and of OPG showed similar cohort specific effects. Still both factors were no surrogates for each other but contributed independent prognostic value in multivariate analyses. Thus, both OPG and PgR are independently associated with good prognosis in ER positive breast cancer. However both markers share common cohort specific differences in contrast to proliferation markers as Ki67 which may be based on the underlying biology.

Topics: Biomarkers, Tumor; Breast; Breast Neoplasms; Female; Humans; Middle Aged; Osteoprotegerin; Prognosis; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Estrogen; Receptors, Progesterone; RNA, Messenger; Transcriptome

Disbalance of bone homeostasis, associated with malfunctioning of RANK/RANKL/OPG system underlies the oncological processes such as the destruction of bone, metastasis development, tumor progression. Pathological activity of system was described in such conditions, as breast cancer, prostate cancer, multiple myeloma, squamous cell carcinoma, Hodgkin's disease, and also metastasis in bones from lung cancer and other malignant diseases. In the literature, there is evidence of involvement of RANK/RANKL/OPG system in the pathogenesis of bone tumors (osteosarcoma, giant cell tumor of bone, chondroblastoma). Experimental data show that RANKL inhibitors can play a role in reducing tumor-induced lesions of bone in multiple myeloma, breast cancer, prostate cancer and lung cancer. Also this review presents data from clinical studies of the drug efficacy targeted on RANK/RANKL/OPG system and results of authors' study of the levels of this system's components and proinflammatory cytokines in blood serum of primary bone sarcoma patients.

Topics: Animals; Antineoplastic Agents; Bone Neoplasms; Breast Neoplasms; Cytokines; Female; Humans; Lung Neoplasms; Male; Multiple Myeloma; Osteoprotegerin; Prostatic Neoplasms; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Signal Transduction

Bone homeostasis is maintained by the remodelling of bone which depends on a balance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. Malignant bone lesions are very common in patients with cancer; whether they result from a tumor in bone (giant cell tumour of bone, osteosarcoma, multiple myeloma...) or they are bony metastases from advanced cancers of which the most osteotropic are breast and prostate cancer. Malignant cells within the bone disrupt the normal bone remodelling process, leading to increased bone destruction and occurence of pathological fractures. Receptor activator of NF-kB (RANK) and its ligand (RANKL) play a pivotal role in the regulation of bone remodelling; by binding to RANK, RANKL stimulates osteoclastogenesis and bone resorption, whereas its cognate decoy receptor osteoprotegerin (OPG) blocks this process by interacting with RANKL. Tumour cells produce different factors that manipulate the RANK/RANKL/OPG pathway in order to stimulate bone destruction. Furthermore, pending on the tumour type, RANKL plays a role in the migration, invasion and proliferation of malignant cells within the bone, while OPG increases survival of tumour cells. Inhibition of RANK/RANKL system may therefore offer new therapeutic perspectives for the treatment of primitive and secondary bone cancers.

Topics: Bone Neoplasms; Bone Remodeling; Bone Resorption; Breast Neoplasms; Female; Homeostasis; Humans; Male; Multiple Myeloma; Neoplasm Proteins; Osteoprotegerin; Prostatic Neoplasms; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B

Cancers which damage the human skeleton include multiple myeloma, where the primary tumour colonises bone directly, or breast and prostate cancer, where malignant cells travel from the primary tumour to form clonal outgrowths within the bone. Owing to the interaction of tumour cells with those normally found in the bone microenvironment, such as osteoclasts and osteoblasts, these cancers affect the closely linked processes of bone formation and resorption. As a result, these twin processes contribute to the clinical manifestations of cancer metastasis, including bone pain and pathological fractures. A critical component of physiologically normal bone remodelling, the RANK/RANKL/OPG pathway, has been implicated in the formation of osteolytic, and possibly osteoblastic, lesions, which characterise the bone disease associated with these malignancies. In these cancers that affect the skeleton in this way the abnormally regulated RANK/RANKL system appears to be the final effector pathway. As a result, there has been much research focused upon targeting these molecules using OPG constructs, peptidomimetics, soluble receptor constructs and antibodies to RANKL, in pre-clinical studies. The success of these studies has paved the way for a clinical programme, the success of which is likely to lead to a new therapeutic approach to treating cancers that develop in the skeleton.

Topics: Animals; Bone Resorption; Breast Neoplasms; Female; Humans; Male; Models, Biological; Multiple Myeloma; Neoplasms; Osteoprotegerin; Prostatic Neoplasms; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Signal Transduction; TNF-Related Apoptosis-Inducing 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

Skeletal-related complications occur commonly in many solid tumors including breast, prostate and lung cancer as well as multiple myeloma. In addition, malignancies and their associated treatment may result in bone loss or osteoporosis. This review will focus solely on recent data associated with metastatic bone disease with a focus on breast cancer, prostate cancer and multiple myeloma. Bone loss or osteoporosis associated with cancer will be covered in a separate article in this issue.. Recent progress in understanding the pathophysiology of bone metastases has pointed to several novel pathways: transforming growth factor beta; receptor activator of nuclear factor beta ligand and osteoprotegerin; and Wnt signaling pathways and associated factors such as dickkopf-1 and endothelin-1.. The identification of new pathways is important in metastatic bone disease from cancer and has allowed for the development of novel therapeutics aimed at preventing the devastating complications of bone metastases. Bisphosphonates remain the predominant therapy in use for the treatment and prevention of skeletal-related adverse effects from cancer.

Topics: Bone Density Conservation Agents; Bone Neoplasms; Breast Neoplasms; Diphosphonates; Endothelin-1; Female; Humans; Intercellular Signaling Peptides and Proteins; Male; Multiple Myeloma; Osteoprotegerin; Prostatic Neoplasms; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Signal Transduction; Transforming Growth Factor beta; Wnt Proteins

Metastasis to the skeleton occurs in approximately 75% of patients with advanced breast cancer. It is the cause of much morbidity and decreased quality of life in these patients.. Zoledronic acid (Zoledronate) is a new, potent third-generation bisphosphonate that has recently been approved by the U.S. Food and Drug Administration for the treatment of both osteolytic and osteoblastic bone metastases. A Phase 3 clinical trial comparing the efficacy of Zoledronic acid 4 and 8 mg compared with Pamidronate 90 mg every 3-4 weeks has been completed.. A 15 minute infusion of 4 mg of zoledronic acid is at least as effective as 90 mg of pamidronate in preventing skeletal complications in patients with metastatic breast cancer.. Bisphosphonate treatment can delay skeletal events in patients with breast carcinoma that is metastatic to bone. Future trials will build on the results achieved with bisphosphonate treatment.

Topics: Administration, Oral; Bone Neoplasms; Breast Neoplasms; Diphosphonates; Female; Forecasting; Glycoproteins; Humans; Imidazoles; Infusions, Intravenous; Osteoprotegerin; Pamidronate; Randomized Controlled Trials as Topic; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Zoledronic Acid

Standard management of breast cancer metastatic to bone includes systemic chemotherapy and, if applicable, hormone therapy, as well as radiotherapy for control of pain or prevention of pathologic fractures. In addition, orthopedic surgical procedures are used to prevent or correct pathologic fractures in weight-bearing areas of the osseous skeleton. Inhibitors of osteoclast function, including bisphosphonates and gallium nitrate, have been shown in clinical trials to decrease bone-related complications. Consequently, bisphosphonates have become an integral part of the management of bone metastases from breast cancer. Improved understanding of the biology of osteoclastogenesis led to the identification of osteoprotegerin as a critical modulator of osteoclast activity. The clinical evaluation of several osteoprotegerin preparations has shown therapeutic effects as measured by significant reductions in biochemical markers of bone resorption. Monoclonal antibodies to RANK ligand and parathyroid hormone-related protein, as well as Src kinase inhibitors, are also currently under clinical evaluation.

Topics: Antineoplastic Agents; Bone Neoplasms; Bone Resorption; Breast Neoplasms; Carrier Proteins; Cytokines; Enzyme Inhibitors; Glycoproteins; Humans; Ligands; Membrane Glycoproteins; Osteoclasts; Osteoprotegerin; Radiopharmaceuticals; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptor, Parathyroid Hormone, Type 1; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Signal Transduction; src-Family Kinases

The most common human cancers --lung, breast and prostate -- have a great avidity for bone, leading to painful and untreatable consequences. What makes some cancers, but not others, metastasize to bone, and how do they alter its physiology? Some of the molecular mechanisms that are responsible have recently been identified, and provide new molecular targets for drug development.

Topics: Bone Neoplasms; Breast Neoplasms; Calcium; Carrier Proteins; Diphosphonates; Female; Glycoproteins; Humans; Lung Neoplasms; Male; Membrane Glycoproteins; Osteoprotegerin; Prostatic Neoplasms; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Vitamin D

The receptor activator of nuclear factor kappa B (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) axis may contribute to the development of bone metastases (BM). We studied gene expression in this pathway in primary breast cancer (BC) to determine correlations with clinical characteristics and outcomes in the neoadjuvant I-SPY1 study.. We evaluated RANK/RANKL/OPG expression using expression microarrays in I-SPY1 (n = 149). Associations with clinical features were determined using t test and ANOVA. Associations between biomarker high versus low groups (dichotomized at an optimal cutpoint) and recurrence-free survival (RFS) were evaluated using the log-rank test and in a multivariate Cox proportional hazard model. A pooled external neoadjuvant cohort with gene expression data (GSE25066) (Hatzis et al. in JAMA 305(18):1873-1881, 30) (n = 425) was used for validation. Associations with site-specific relapse were evaluated using the t-test and multivariate logistic regression adjusting for hormone receptor (HR) status.. RANK was significantly higher in HR negative versus HR positive (p = 0.027), in basal versus non-basal disease (p = 0.004), and in those achieving pathologic complete response (p = 0.038); the associations with HR negative and basal BC were also significant in GSE25066. In both datasets, higher RANK associated with significantly worse RFS (I-SPY1: p = 0.045, GSE25066: p = 0.044). However, this association did not remain significant after adjusting for HR status. In I-SPY1 patients with recurrence, higher RANK correlated with BM versus non-BM (p = 0.045), even after adjusting for HR status (p = 0.035).. RANK is increased in HR negative and basal BC, and correlates with worse RFS and risk of BM. The RANK pathway is a potential therapeutic target in BC.

Topics: Biomarkers, Tumor; Bone Neoplasms; Breast Neoplasms; Chemotherapy, Adjuvant; Disease-Free Survival; Female; Gene Expression Profiling; Humans; Logistic Models; Mastectomy; Middle Aged; Multivariate Analysis; Neoadjuvant Therapy; Neoplasm Recurrence, Local; Oligonucleotide Array Sequence Analysis; Osteoprotegerin; Proportional Hazards Models; Radiotherapy, Adjuvant; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Risk Assessment; Risk Factors; Time Factors; Treatment Outcome

Patients with solid cancer frequently develop bone metastases (BM). Zoledronic acid (Zometa®, ZA), routinely used to treat patients with BM, acts on osteoclasts and also has antitumor properties. We aimed to assess the effect of ZA over time in novel bone turnover markers (RANK/receptor activator of nuclear factor-k B ligand (RANK-L)/ Osteoprotegerin (OPG)) and to correlate these with serum N-terminal telopeptide (NTX). The study prospectively evaluated levels of RANK, RANK-L and OPG transcripts by real-time PCR and NTX expression by ELISA in the peripheral blood of 49 consecutive patients with advanced breast, lung or prostate cancer. All patients received the standard ZA schedule and were monitored for 12 months. Median baseline values of RANK, RANK-L and OPG were 78.28 (range 7.34-620.64), 319.06 (21.42-1884.41) and 1.52 (0.10-58.02), respectively. At 12 months, the median RANK-L value had decreased by 22% with respect to the baseline, whereas median OPG levels had increased by about 96%. Consequently, the RANK-L/OPG ratio decreased by 56% from the baseline. Median serum NTX levels decreased over the 12-month period, reaching statistical significance (p < 0.0001). Our results would seem to indicate that ZA modulates RANK, RANK-L and OPG expression, thus decreasing osteoclast activity.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Biomarkers, Tumor; Bone Neoplasms; Breast Neoplasms; Collagen Type I; Diphosphonates; Female; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Male; Middle Aged; Osteoprotegerin; Peptides; Prospective Studies; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Signal Transduction; Zoledronic Acid

Metastasis to the skeleton occurs in approximately 75% of patients with advanced breast cancer. It is the cause of much morbidity and decreased quality of life in these patients.. Zoledronic acid (Zoledronate) is a new, potent third-generation bisphosphonate that has recently been approved by the U.S. Food and Drug Administration for the treatment of both osteolytic and osteoblastic bone metastases. A Phase 3 clinical trial comparing the efficacy of Zoledronic acid 4 and 8 mg compared with Pamidronate 90 mg every 3-4 weeks has been completed.. A 15 minute infusion of 4 mg of zoledronic acid is at least as effective as 90 mg of pamidronate in preventing skeletal complications in patients with metastatic breast cancer.. Bisphosphonate treatment can delay skeletal events in patients with breast carcinoma that is metastatic to bone. Future trials will build on the results achieved with bisphosphonate treatment.

Topics: Administration, Oral; Bone Neoplasms; Breast Neoplasms; Diphosphonates; Female; Forecasting; Glycoproteins; Humans; Imidazoles; Infusions, Intravenous; Osteoprotegerin; Pamidronate; Randomized Controlled Trials as Topic; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Zoledronic Acid

Osteoprotegerin (OPG) is a decoy receptor for OPG ligand (OPGL), or receptor activator of NF-kappaB ligand (RANKL). RANKL/RANK interaction is important in terminal differentiation and activation of osteoclasts. In binding to RANKL, OPG blocks differentiation and activation of osteoclasts. AMGN-0007 is a recombinant OPG construct developed as a potential therapeutic agent in the treatment of bone disease.. A randomized, double-blind, double-dummy, active-controlled, single-dose, dose escalation study was conducted to determine the safety and effect on bone resorption of AMGN-0007 in patients with multiple myeloma (n = 28) or breast carcinoma (n = 26) with radiologically confirmed lytic bone lesions. Patients were randomized (3:1 ratio) to receive a single dose of either AMGN-0007 (subcutaneously [SC]) or pamidronate (90 mg intravenously) and were followed for 56 days. Medications or other diseases affecting bone metabolism and chemotherapy within 28 days of dosing were exclusion criteria. Biologic activity of AMGN-0007 was assessed by measurement of the surrogate marker of bone resorption, urinary N-telopeptide of collagen (NTX).. AMGN-0007 caused a rapid, sustained, dose-dependent decrease in NTX/creatinine levels, which was at least comparable to the profile observed with pamidronate. Four serious adverse events were reported, three in breast carcinoma patients: a fracture in the left femur (pamidronate, considered unrelated), extreme fatigue (0.3 mg/kg AMGN-0007, considered unrelated), and congestive heart failure (1.0 mg/kg AMGN-0007, considered by the investigator to be probably related to doxorubicin and radiation therapy); one event occurred in a multiple myeloma patient: Herpes zoster (pamidronate, considered unrelated). Two multiple myeloma patients (1.0 mg/kg AMGN-0007) had albumin-adjusted serum calcium levels of 1.9 mmol/L on Day 8 but without clinical symptoms.. A single SC dose of AMGN-0007 suppressed bone resorption as indicated by a rapid, sustained, and profound decrease of urinary NTX/creatinine in multiple myeloma and breast carcinoma patients. Changes were comparable to those with pamidronate. AMGN-0007 was well tolerated.

Topics: Antineoplastic Agents; Bone Neoplasms; Bone Resorption; Breast Neoplasms; Diphosphonates; Dose-Response Relationship, Drug; Double-Blind Method; Female; Glycoproteins; Humans; Male; Middle Aged; Multiple Myeloma; Osteoprotegerin; Pamidronate; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Treatment Outcome

Pre-metastatic niche is a location where cancer cells, separating from a primary tumor, find "fertile soil" for growth and proliferation, ensuring successful metastasis. Exosomal miRNAs of breast cancer are known to enter the bone and degrade it, which facilitates cancer cells invasion into the bone interior and ensures its successful colonization. In this paper, we use a mathematical model to first describe, in health, the continuous remodeling of the bone by bone-forming osteoblasts, bone-resorbing osteoclasts and the RANKL-OPG-RANK signaling system, which keeps the balance between bone formation and bone resorption. We next demonstrate how breast cancer exosomal miRNAs disrupt this balance, either by increasing or by decreasing the ratio of osteoclasts/osteoblasts, which results in abnormal high bone resorption or abnormal high bone forming, respectively, and in bone weakening in both cases. Finally we consider the case of abnormally high resorption and evaluate the effect of drugs, which may increase bone density to normal level, thus protecting the bone from invasion by cancer cells.

Topics: Bone Resorption; Breast Neoplasms; Female; Humans; Mathematical Concepts; MicroRNAs; Models, Biological; Osteoblasts; Osteoclasts; Osteoprotegerin

Experimental studies suggest a role for osteoprotegerin (OPG) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in mammary tumor development and progression. These biomarkers have been minimally investigated with respect to outcomes in breast cancer patients.. OPG and TRAIL were evaluated in blood samples collected from 2459 breast cancer patients enrolled in the MARIE study, a prospective population-based patient cohort, at median of 129 days after diagnosis. Participants were between ages 50 and 74 at diagnosis and recruited from 2002 to 2005 in two regions of Germany. Follow-up for recurrence and mortality was conducted through June 2015. Delayed-entry Cox proportional hazards regression was used to assess associations between OPG and TRAIL with all-cause and breast cancer-specific mortality, and recurrence, both overall and by tumor hormone receptor status.. Median follow-up time was 11.7 years, with 485 deaths reported (277 breast cancer-specific). Higher OPG concentrations were associated with a higher risk of all-cause mortality (hazard ratio for 1-unit log2-transformed concentration (HR. Higher circulating OPG may be a biomarker of a higher risk of poor outcome among women diagnosed with ER- breast cancer. Further mechanistic studies are warranted.

Topics: Aged; Biomarkers; Breast Neoplasms; Female; Hormones; Humans; Ligands; Middle Aged; Osteoprotegerin; Prospective Studies; TNF-Related Apoptosis-Inducing Ligand

Genotoxic agents are widely used anti-cancer therapies because of their ability to interfere with highly proliferative cells. An important outcome of these interventions is the induction of a state of permanent arrest also known as cellular senescence. However, senescent cancer cells are characterized by genomic instability and are at risk of escaping the growth arrest to eventually facilitate cancer relapse. The tumor necrosis factor related apoptosis inducing ligand (TRAIL) signals extrinsic apoptosis via Death Receptors (DR) 4 and 5, while Decoy Receptors (DcR) 1 and 2, and Osteoprotegerin (OPG) are homologous to death receptors but incapable of transducing an apoptotic signal. The use of recombinant TRAIL as an anti-cancer strategy in combination with chemotherapy is currently in development, and a major question remains whether senescent cancer cells respond to TRAIL. Here, we show variable sensitivity of cancer cells to TRAIL after senescence induction, and upregulation of both pro-apoptotic and anti-apoptotic receptors in therapy-induced senescent cancer cells. A DR5-selective TRAIL variant (DHER), unable to bind to DcR1 or OPG, was more effective in inducing apoptosis of senescent cancer cells compared to wild-type TRAIL. Importantly, no apoptosis induction was observed in non-cancerous cells, even at the highest concentrations tested. Our results suggest that targeting DR5 can serve as a novel therapeutic strategy for the elimination of therapy-induced senescent cancer cells.

Topics: Apoptosis; Breast Neoplasms; Cellular Senescence; Doxorubicin; Female; Gene Expression Regulation, Neoplastic; Genomic Instability; GPI-Linked Proteins; Humans; MCF-7 Cells; Osteoprotegerin; Ovarian Neoplasms; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor, Member 10c; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor Decoy Receptors

Hormones impact breast tissue proliferation. Studies investigating the associations of circulating hormone levels with mammographic breast density have reported conflicting results. Due to the limited number of studies, we investigated the associations of hormone gene expression as well as their downstream mediators within the plasma with mammographic breast density in postmenopausal women.. We recruited postmenopausal women at their annual screening mammogram at Washington University School of Medicine, St. Louis. We used the NanoString nCounter platform to quantify gene expression of hormones (prolactin, progesterone receptor (PGR), estrogen receptor 1 (ESR1), signal transducer and activator of transcription (STAT1 and STAT5), and receptor activator of nuclear factor-kB (RANK) pathway markers (RANK, RANKL, osteoprotegerin, TNFRSF18, and TNFRSF13B) in plasma. We used Volpara to measure volumetric percent density, dense volume, and non-dense volume. Linear regression models, adjusted for confounders, were used to evaluate associations between gene expression (linear fold change) and mammographic breast density.. One unit increase in ESR1, RANK, and TNFRSF18 gene expression was associated with 8% (95% CI 0-15%, p value = 0.05), 10% (95% CI 0-20%, p value = 0.04) and % (95% CI 0-9%, p value = 0.04) higher volumetric percent density, respectively. There were no associations between gene expression of other markers and volumetric percent density. One unit increase in osteoprotegerin and PGR gene expression was associated with 12% (95% CI 4-19%, p value = 0.003) and 7% (95% CI 0-13%, p value = 0.04) lower non-dense volume, respectively.. These findings provide new insight on the associations of plasma hormonal and RANK pathway gene expression with mammographic breast density in postmenopausal women and require confirmation in other studies.

Topics: Breast Density; Breast Neoplasms; Female; Gene Expression; Hormones; Humans; Mammography; Osteoprotegerin; Postmenopause; Receptor Activator of Nuclear Factor-kappa B; Risk Factors

Breast carcinomas (BC) with osteoclast-like giant cells (OGC) are rare. Despite their distinct stromal features, their molecular characteristics remain unknown. Here, we report comprehensive clinico-pathological and molecular findings for 27 patients diagnosed with BC-OGC at Institut Curie between 2000 and 2021. Seventeen (63%) cases were invasive carcinomas of no special type (IC NST) with OGC (OGC-IC NST), four (15%) were mixed or multifocal cases with and without OGC (OGC-Mixed), and six (22%) were metaplastic carcinomas with OGC (OGC-MC). All OGC-IC NST and OGC-Mixed cases were ER+ HER2- tumors (most being luminal A based on transcriptomic subtyping, when available), while all OGC-MC were triple-negative. The median age at diagnosis was 46, 45 and 62 years for OGC-IC NST, OGC-Mixed and OGC-MC, respectively. Three patients developed distant metastases (one OGC-IC NST, two OGC-Mixed), one of whom died of metastatic disease (OGC-Mixed), and one other patient died of locally advanced disease (OGC-MC). Histopathological evaluation comparing 13 OGC-IC NST and 19 control IC NST without OGC confirmed that OGC-IC NST showed significantly higher density of vessels (by CD34 immunohistochemistry (IHC)), iron deposits (Perls stain), and CD68 and CD163-positive cell infiltrates. Genomic findings for nine OGC-IC NST and four OGC-MC were consistent with the underlying histologic subtype, including activating alterations of the PI3K/AKT/mTOR pathway in 7/13 cases. Using RNA-seq data, differential gene expression analysis between OGC-IC NST (n = 7) and control IC NST without OGC (n = 7) revealed significant overexpression of TNFSF11 (RANK-L), TNFRSF11A (RANK), CSF1 (M-CSF), CSF1R, and genes encoding osteoclastic enzymes (MMP9, ACP5, CTSK, CTSB) in OGC-IC NST, while OPG (osteoprotegerin) was underexpressed. We also confirmed for the first time RANK-L expression in BC with OGC by IHC (seen in 15 out of 16 cases, and only in 2 of 16 controls without OGC). These findings could offer a rationale for further investigating RANK-L as a therapeutic target in BC with OGC.

Topics: Breast Neoplasms; Carcinoma; Female; Giant Cells; Humans; Iron; Macrophage Colony-Stimulating Factor; Matrix Metalloproteinase 9; Osteoclasts; Osteoprotegerin; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RANK Ligand; TOR Serine-Threonine Kinases

Breast cancer results in a three- to four-fold increased risk of venous thromboembolism (VTE), which is associated with reduced patient survival. Despite this, the mechanisms underpinning breast cancer-associated thrombosis remain poorly defined. Tumor cells can trigger endothelial cell (EC) activation resulting in increased von Willebrand factor (VWF) secretion. Importantly, elevated plasma VWF levels constitute an independent biomarker for VTE risk. Moreover, in a model of melanoma, treatment with low molecular weight heparin (LMWH) negatively regulated VWF secretion and attenuated tumor metastasis.. To investigate the role of VWF in breast cancer metastasis and examine the effect of LMWH in modulating EC activation and breast tumor transmigration.. von Willebrand factor levels were measured by ELISA. Primary ECs were used to assess tumor-induced activation, angiogenesis, tumor adhesion, and transendothelial migration.. Patients with metastatic breast cancer have markedly elevated plasma VWF:Ag levels that also correlate with poorer survival. MDA-MB-231 and MCF-7 breast cancer cells induce secretion of VWF, angiopoietin-2, and osteoprotegerin from ECs, which is further enhanced by the presence of platelets. Vascular endothelial growth factor-A (VEGF-A) plays an important role in modulating breast cancer-induced VWF release. Moreover, VEGF-A from breast tumor cells also contributes to a pro-angiogenic effect on ECs. VWF multimers secreted from ECs, in response to tumor-VEGF-A, mediate adhesion of breast tumor cells along the endothelium. LMWH inhibits VWF-breast tumor adhesion and transendothelial migration. Our findings highlight the significant crosstalk between tumor cells and the endothelium including increased VWF secretion which may contribute to tumor metastasis.

Topics: Angiopoietin-2; Breast Neoplasms; Endothelial Cells; Female; Heparin, Low-Molecular-Weight; Humans; Osteoprotegerin; Transendothelial and Transepithelial Migration; Vascular Endothelial Growth Factor A; Venous Thromboembolism; von Willebrand Factor

The receptor activator NF-κB ligand (RANKL) and Osteoprotegrin (OPG) single nucleotide polymorphisms (SNPs) have been associated with the risk of breast cancer to bone metastasis. This study was designed to investigate the association of RANKL and OPG gene polymorphisms with breast to bone metastasis in Pashtun population of Khyber Pakhtunkhwa, Pakistan.. A total of 215 participants were enrolled containing 106 breast cancer patients, 58 breast to bone metastasis and 51 age and gender matched healthy controls. RANKL (rs9533156) and OPG (rs2073618, rs3102735) polymorphisms were genotyped in genomic DNA, using Tetra-ARMS PCR protocol. The results were analyzed among the three groups and P-value less then 0.05 were considered statistically significant.. Our results displayed significant association of OPG (rs3102735) risk allele and corresponding genotypes in breast cancer vs healthy controls, bone metastasis vs healthy controls and breast cancer vs breast to bone metastasis as a disease risk. However, there was no association observed for OPG (rs2073618) risk allele and corresponding genotypes with the diseases risk. Similarly, RANKL (rs9533156) risk allele and corresponding genotypes in breast cancer vs healthy controls, bone metastasis vs healthy controls and breast cancer vs breast to bone metastasis exhibited significant association except for the risk allele carrying genotypes in breast to bone metastasis.. OPG (rs3102735) and RANKL (rs9533156) exhibited significant association with breast to bone metastasis while OPG (rs2073618) didn't show significant association with breast to bone metastasis in Pashtun population of Pakistan. However, this study unlocks more questions to investigate the exact scenario of genetic predisposition of breast to bone metastasis.

Topics: Breast Neoplasms; Female; Humans; Ligands; NF-kappa B; Osteoprotegerin; Pakistan; Polymorphism, Single Nucleotide; RANK Ligand

Breast carcinomas are composed of cancer cells surrounded by various types of non-cancer cells such as fibroblasts. While active cancer-associated fibroblasts (CAFs) support tumor initiation and progression, quiescent breast stromal fibroblasts (BSFs) inhibit these effects through various cytokines such as osteoprotegerin (OPG). We showed here that OPG is upregulated in CAFs as compared to their adjacent normal tumor counterpart fibroblasts. Interestingly, breast cancer cells can upregulate OPG in BSFs in an IL-6-dependent manner through the IL-6/STAT3 pathway. When upregulated by ectopic expression, OPG activated BSFs through the NF-κB/STAT3/AUF1 signaling pathway and promoted their paracrine pro-carcinogenic effects in an IL-6-dependent manner. In addition, this increase in the OPG level enhanced the potential of BSFs to promote the growth of humanized orthotopic tumors in mice. However, specific OPG knock-down suppressed active CAFs and their paracrine pro-carcinogenic effects. Similar effects were observed when CAF cells were exposed to the pure recombinant OPG (rOPG) protein. Together, these findings show the importance of OPG in the activation of stromal fibroblasts and the possible use of rOPG or inhibitors of the endogenous protein to target CAFs as precision cancer therapeutics.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Female; Fibroblasts; Humans; Interleukin-6; Mice; Osteoprotegerin; Signal Transduction; STAT3 Transcription Factor; Up-Regulation

Breast cancer represents one of the most popular kinds of cancer worldwide. During the early stages of the disease, the level of Osteoprotegerin remained within normal limits, showing that the bone was not being damaged to get calcium due to an increase in parathyroid hormone. The current study aimed to assess a number of biochemical variables in a group of women with malignant breast cancer who had reached menopause (less than 45 years old). One hundred thirty women were randomly divided into three groups as follows. The first group (G1) is made up of women who have never had breast cancer or any other disease, and their number (40) corresponds to the same age range (below menopause) as the control group. The second group (G2) comprises women diagnosed with breast cancer at an early stage whose numbers were relatively low (45). The third group (G3) included women of the same age who received one or two doses of chemotherapy and whose total number was (45) over the same period. The variables studied include Vitamin D, Parathyroid Hormone, Osteoprotegerin, blood calcium, and urine calcium, all of that are thought to play a role in the progress of the disease. Vitamin D levels were extremely low in the second group (G2), while they were slightly higher in the third group (G3) but remained extremely low. The first group (G1) maintained parameters within acceptable limits. There was a significant difference between the two breast cancer groups (9.38 1.43) and (4.98 1.67) when compared to the control group (20.04 2.80). (G1). The two breast cancer groups (G2) and (G3) had higher parathyroid hormone levels than the control group (G1), and there was a significant difference between the two breast cancer groups (136.52 58.56) (G3) and (G2) (167.79 35.21) compared to the control group (68.52 20.44) (G1). There was no significant difference in Osteoprotegerin levels between the two breast cancer groups (313.38 109.02) (G3) and (315.0 123.98) (G2) compared to the control group (G1) (324.11 104.73). The three groups' blood calcium levels were all within normal ranges, and there was no statistically significant difference between them (9.21 0.45), (9.23 0.38), and (9.23 0.38) (G3) (9.28 0.43). (G1), but urine-calcium levels were lower in both groups of breast cancer patients compared to the control group, and there was a significant difference between the two breast cancer groups (63.96 15.66) (G3) and (68.42 14.05) (G2) compared to the control group (213.77 63.94) (G

Topics: Breast Neoplasms; Calcium; Female; Humans; Osteoprotegerin; Parathyroid Hormone; Vitamin D

Emerging evidence suggests that the progesterone-mediated receptor activator of nuclear factor κB (RANK)/soluble RANK ligand (sRANKL)/osteoprotegerin (OPG) pathway plays an important role in mammary carcinogenesis and is hyperactivated in germline (g)BRCA1/2 mutation carriers. We analyzed the effects of a 3-month intensive lifestyle intervention within the LIBRE-1 study on the serum levels of OPG and sRANKL and hypothesized that the intervention program provides a beneficial impact on the biomarkers by increasing OPG and reducing sRANKL serum concentrations.. Serum levels of OPG and sRANKL of 49 gBRCA1/2 mutation carriers were quantified using enzyme-linked immunosorbent assays. We used previously collected blood samples from participants of the prospective LIBRE-1 study, who were randomized into an intervention group (IG), increasing physical activity and adherence to the Mediterranean diet (MedD) through supervised sessions from study entry to the first study visit after 3 months and a usual-care control group (CG). Differences in biomarker levels before and after the 3-month intervention were tested within and between study groups.. Findings from this pilot study seem to confirm our hypothesis by showing an increase in OPG and decrease in sRANKL over a 3-month lifestyle intervention and suggest that increased physical activity and adherence to the MedD are potent modulators of the biomarkers OPG and potentially sRANKL.

Topics: BRCA1 Protein; BRCA2 Protein; Breast Neoplasms; Diet, Mediterranean; Exercise; Female; Humans; Life Style; Mutation; Osteoprotegerin; Pilot Projects; Prospective Studies; Randomized Controlled Trials as Topic; RANK Ligand

Normal breast fibroblasts (NBFs) support and maintain the architecture of the organ, and can also suppress tumorigenesis. However, the mechanisms involved are not fully understood. We have shown here that NBFs suppress breast carcinogenesis through secretion of osteoprotegerin (OPG), a soluble decoy receptor for the Receptor Activator of NF-κB ligand (RANKL). Indeed, NBFs and human recombinant OPG (rOPG), suppressed breast cancer cells proliferation and motility through inhibition of the epithelial-to-mesenchymal transition (EMT) process both in vitro and in vivo. Additionally, rOPG inhibited the IL-6/STAT3 and NF-κB pathways as well as the OPG gene, which turned out to be STAT3-regulated. This was confirmed using denosumab, a RANKL-targeted antibody, which also inhibited NF-κB, down-regulated OPG and repressed EMT in breast cancer cells grown in 2D and 3D. Importantly, both rOPG and denosumab targeted cancer stem cells (CSCs). This was mediated through inhibition of the CSC-related pathway β-catenin. Moreover, rOPG reduced tumor growth and inhibited breast CSC biomarkers in orthotopic humanized breast tumors. Therefore, normal mammary fibroblasts can suppress carcinogenesis through OPG, which constitutes great potential as preventive and/or therapeutic molecule for breast carcinomas.

Topics: Antibodies; Anticarcinogenic Agents; beta Catenin; Breast Neoplasms; Carcinogenesis; Cell Movement; Cell Proliferation; Denosumab; Epithelial-Mesenchymal Transition; Female; Fibroblasts; Gene Expression Regulation, Neoplastic; Humans; Interleukin-6; Neoplastic Stem Cells; NF-kappa B; Osteoprotegerin; RANK Ligand; Recombinant Proteins; STAT3 Transcription Factor

Searching for sensitive, minimally invasive biomarkers that represent tumor-associated changes in the peripheral blood might enable the early diagnosis of breast cancer (BC) and monitoring of tumor progression.. Herein, we investigated the association of some circulating biomarkers with the risk of metastasis. In the current study, 115 BC patients which were subdivided into two groups: nonmetastatic breast cancer patients (NMBC) (n=83) and metastatic breast cancer patients (MBC) (n=32), and 79 apparently healthy controls were recruited. Serum protein levels of lysosomal protein transmembrane 4 beta (LAPTM4B), receptor activator of nuclear factor-kappa b (NF-Kb) ligand (RANKL), osteoprotegerin (OPG), vitamin D (VIT D), chitinase-3-like protein 1 (also known as YKL-40), and sirtuin 1 (SIRT1) were assessed in blood samples using ELISA technique.. The results showed that RANKL and OPG had the highest diagnostic potential for MBC detection, with area under the curve values of 0.97 and 0.94, respectively. Moreover, logistic regression analysis showed that RANKL had the highest differentiation power in the discrimination of MBC from NMBC.. The study highlighted that measuring RANKL and OPG may be helpful in the early detection of metastasis in Egyptian patients with BC.

Topics: Adult; Aged; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Case-Control Studies; Egypt; Female; Follow-Up Studies; Humans; Lymphatic Metastasis; Middle Aged; Neoplasm Invasiveness; Osteoprotegerin; Prognosis; RANK Ligand; ROC Curve; Young Adult

Emerging evidence supports a role of the receptor activator of NF-κB (RANK) pathway in normal mammary gland development and breast carcinogenesis. Osteoprotegerin (OPG) is the endogenous decoy receptor for RANK-ligand (RANKL), which inhibits RANK-signaling. Whether OPG may be a biomarker of breast cancer risk remains unclear.. We evaluated the association between plasma OPG and breast cancer risk in a case (. Overall, there was no substantial evidence for an association between plasma OPG levels and breast cancer risk, although the point estimate for the highest (vs. lowest) quartile was below 1 (OR = 0.78; 95% CI, 0.46-1.33;. Findings from this prospective study do not provide substantial evidence for an association between circulating OPG and breast cancer risk among premenopausal women; however, we were underpowered in stratified analyses.. Results do not provide strong evidence for OPG as a potential biomarker of breast cancer risk among premenopausal women.

Topics: Adult; Breast Neoplasms; Case-Control Studies; Female; Humans; Middle Aged; Nurses; Osteoprotegerin; Premenopause; Risk Factors

Chemerin is secreted as prochemerin from various cell types and then cleaved into the bioactive isoform by specific proteases. In various cancer types, chemerin exhibits pro- or antitumor effects. In the present study, chemerin treatment significantly inhibited the viability and invasion of breast cancer cells in the absence or presence of transforming growth factor (TGF)-β and insulin-like growth factor (IGF)-1. The expression levels of E-cadherin and vimentin were reduced in chemerin-treated breast cancer cells. However, chemerin treatment recovered the reduced E-cadherin expression level in breast cancer cells treated with TGF-β or IGF-1. Chemerin treatment inhibited nuclear β-catenin levels in breast cancer cells stimulated with or without TGF-β or IGF-1. In addition, chemerin treatment blocked the increase in the receptor activator of nuclear factor kappa-Β ligand (RANKL)/osteoprotegerin (OPG) ratio in osteoblastic cells exposed to metastatic breast cancer cell-derived conditioned medium. Chemerin treatment inhibited RANKL-induced osteoclast formation and bone resorption by reducing the secretion of matrix metalloproteinase (MMP)-2, MMP-9, and cathepsin K. Intraperitoneal administration of chemerin inhibited tumor growth in MCF-7 breast cancer cell-injected mice and reduced the development of osteolytic lesions resulting from intratibial inoculation of MDA-MB-231 cells. Taken together, chemerin inhibits the growth and invasion of breast cancer cells and prevents bone loss resulting from breast cancer cells by inhibiting finally osteoclast formation and activity.

Topics: Animals; Antineoplastic Agents; Biomarkers; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chemokines; Disease Models, Animal; Female; Gene Expression; Humans; Immunophenotyping; Mice; Osteoprotegerin; RANK Ligand; Xenograft Model Antitumor Assays

Genetic susceptibility for breast cancer (BC) is still poorly understood. A combination of multiple low-penetrant alleles of cancer-related genes and gene-gene interactions (epistasis) contributes to BC risk. Genetic variants in receptor activator of nuclear factor κB ligand (RANKL), osteoprotegerin (OPG), chitinase-3-like protein 1 (CHI3L1), and vitamin D receptor (VDR) genes are implicated in breast carcinogenesis; however, the influence of their epistatic effects on BC susceptibility has not yet been studied. We investigated the association of single nucleotide polymorphism (SNP)-SNP interactions and haplotypes of 6 SNPs in these 4 genes with the genetic predisposition of BC in Egyptian women.. Data of 115 BC patients and 120 cancer-free controls were studied. Association tests were conducted using logistic regression models.. Individual SNPs showed weak statistical significance with BC susceptibility. The interactions between RANKL-rs9533156 and OPG-rs2073618; OPG-rs2073618 with CHI3L1-rs4950928, VDR-rs2228570 and VDR-rs1544410; OPG-rs2073617 and VDR-rs1544410; VDR-rs2228570 and VDR-rs1544410 were strongly associated with increased BC risk after adjustment for multiple comparisons. No SNPs were in strong linkage disequilibrium. The TCTCTG-rs9533156-rs2073618-rs2073617-rs4950928-rs2228570-rs1544410 haplotype was significantly associated with increased BC risk (adjusted odds ratio = 8.33; 95% confidence interval, 1.32-52.46; P = .025) compared with controls. TCCCTG haplotype stratified BC patients according to estrogen receptor/progesterone receptor status. TCTCTA was positively associated, and TCTCTG and TGTCTG haplotypes inversely correlated with bone metastasis. Bioinformatic analysis revealed 13 proteins commonly interacting with our 4 genes; the most significant was signal transducer and activator of transcription 5B.. Our results suggested that a stronger combined effect of SNPs in RANKL, OPG, CHI3L1, and VDR genes via gene-gene interaction may help predict BC risk and prognosis.

Topics: Adult; Aged; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Case-Control Studies; Chitinase-3-Like Protein 1; Egypt; Female; Follow-Up Studies; Genetic Predisposition to Disease; Humans; Lymphatic Metastasis; Middle Aged; Osteoprotegerin; Polymorphism, Single Nucleotide; Prognosis; RANK Ligand; Receptors, Calcitriol; Retrospective Studies; Risk Factors

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

Bone metastases are common and devastating to cancer patients. Existing treatments do not specifically target the disease sites and are therefore ineffective and systemically toxic. Here we present a new strategy to treat bone metastasis by targeting both the cancer cells ("the seed"), and their surrounding niche ("the soil"), using stem cells engineered to home to the bone metastatic niche and to maximise local delivery of multiple therapeutic factors.. We used mesenchymal stem cells engineered using mRNA to simultaneously express P-selectin glycoprotein ligand-1 (PSGL-1)/Sialyl-Lewis X (SLEX) (homing factors), and modified versions of cytosine deaminase (CD) and osteoprotegerin (OPG) (therapeutic factors) to target and treat breast cancer bone metastases in two mouse models, a xenograft intratibial model and a syngeneic model of spontaneous bone metastasis.. We first confirmed that MSC engineered using mRNA produced functional proteins (PSGL-1/SLEX, CD and OPG) using various in vitro assays. We then demonstrated that mRNA-engineered MSC exhibit enhanced homing to the bone metastatic niche likely through interactions between PSGL-1/SLEX and P-selectin expressed on tumour vasculature. In both the xenograft intratibial model and syngeneic model of spontaneous bone metastasis, engineered MSC can effectively kill tumour cells and preserve bone integrity. The engineered MSC also exhibited minimal toxicity in vivo, compared to its non-targeted chemotherapy counterpart (5-fluorouracil).. Our combinatorial targeting of both the cancer cells and the niche represents a simple, safe and effective way to treat metastatic bone diseases, otherwise difficult to manage with existing strategies. It can also be applied to other cell types (e.g., T cells) and cargos (e.g., genome editing components) to treat a broad range of cancer and other complex diseases. FUND: National Institutes of Health, National Cancer Institute of the National Institutes of Health, Department of Defense, California Institute of Regenerative Medicine, National Science Foundation, Baylx Inc., and Fondation ARC pour la recherche sur le cancer.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Cell Engineering; Cell Line, Tumor; Cytosine Deaminase; Female; Genetic Therapy; Humans; Membrane Glycoproteins; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Osteoprotegerin; P-Selectin; RAW 264.7 Cells; RNA, Messenger; Sialyl Lewis X Antigen; Xenograft Model Antitumor Assays

The bone represents one of the most common sites of metastases in breast cancer. The aim of the current study was to evaluate the diagnostic potential of several circulating markers to detect metastasis to bones in patients with breast cancer.. Receptor activator of Nuclear Factor-kappa β (NF-Kβ) ligand (RANKL), osteoprotegrin (OPG), vitamin D (VIT D), Chitinase-3-like protein 1; also known as YKL-40, topoisomerase IIα (TOPO2a), and human epidermal growth factor receptor 2 (HER2) were measured in blood samples obtained from 122 patients with breast cancer and 25 healthy controls.. All biomarkers were significantly elevated in patients with breast cancer with bone metastasis compared with nonmetastatic patients except YKL-40. RANKL had the highest diagnostic performance for bone metastasis detection with an area under the curve of 97.3, a sensitivity of 85%, and a specificity of 98.6%. Furthermore, logistic regression analysis resulted in a model of RANKL combined with HER2 that had even higher discriminatory power of metastasis to bones than that of RANKL alone. Overall correct classification of the model was 98.9%.. We recommend that measuring RANKL together with HER2 can be routinely applied to allow early detection of bone metastases in patients with breast cancer.

Topics: Adult; Biomarkers, Tumor; Biopsy; Bone Neoplasms; Breast; Breast Neoplasms; Chitinase-3-Like Protein 1; DNA Topoisomerases, Type II; Early Detection of Cancer; Female; Humans; Logistic Models; Mammography; Osteoprotegerin; Poly-ADP-Ribose Binding Proteins; RANK Ligand; Receptor, ErbB-2; Vitamin D

Aromatase inhibitor (AI) therapy is highly efficacious in the treatment of estrogen receptor-positive breast cancer; however, in a subset of patients AI use is discontinued due to drug-induced musculoskeletal adverse events (MS-AE). Several studies have investigated the role of germline single nucleotide polymorphisms (SNPs) on patients' risk of MS-AEs; however, no associations have yet to be validated for translation into clinical practice. This study attempted to replicate SNPs in RANKL ( rs7984870 ) and OPG ( rs2073618 ) on the risk of AI-induced MS-AEs and screen for secondary associations with MS-AE-related treatment discontinuation and serum and urine markers of bone health. Previously reported associations were not replicated with our primary hypothesis, change in MS-AE from baseline to 3 mo; however, patients homozygous for the G allele of rs7984870 in RANKL had lower risk of MS-AE-associated treatment discontinuation in analyses of secondary phenotypes without statistical correction.

Topics: Adult; Aged; Aromatase Inhibitors; Breast Neoplasms; Female; Genetic Predisposition to Disease; Humans; Middle Aged; Osteoprotegerin; Polymorphism, Single Nucleotide; Prospective Studies; RANK Ligand

Mammographic density is a risk factor for breast cancer but the mechanism behind this association is unclear. The receptor activator of nuclear factor κB (RANK)/RANK ligand (RANKL) pathway has been implicated in the development of breast cancer. Given the role of RANK signaling in mammary epithelial cell proliferation, we hypothesized this pathway may also be associated with mammographic density. Osteoprotegerin (OPG), a decoy receptor for RANKL, is known to inhibit RANK signaling. Thus, it is of interest to evaluate whether OPG levels modify breast cancer risk through mammographic density.. We quantified serum OPG levels in 57 premenopausal and 43 postmenopausal women using an enzyme-linked immunosorbent assay (ELISA). Cumulus was used to measure percent density, dense area, and non-dense area for each mammographic image. Subjects were classified into high versus low OPG levels based on the median serum OPG level in the entire cohort (115.1 pg/mL). Multivariate models were used to assess the relationship between serum OPG levels and the measures of mammographic density.. Serum OPG levels were not associated with mammographic density among premenopausal women (P ≥ 0.42). Among postmenopausal women, those with low serum OPG levels had higher mean percent mammographic density (20.9% vs. 13.7%; P = 0.04) and mean dense area (23.4 cm. These findings suggest that low OPG levels may be associated with high mammographic density, particularly in postmenopausal women. Targeting RANK signaling may represent a plausible, non-surgical prevention option for high-risk women with high mammographic density, especially those with low circulating OPG levels.

Topics: Adult; Aged; Breast Density; Breast Neoplasms; Cohort Studies; Female; Humans; Middle Aged; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Risk Factors

Receptor activator of nuclear factor kappa-B (RANK)-signaling is involved in tumor growth and spread in experimental models. Binding of RANK ligand (RANKL) to RANK activates signaling, which is inhibited by osteoprotegerin (OPG). We have previously shown that circulating soluble RANKL (sRANKL) and OPG are associated with breast cancer risk. Here we extend these findings to provide the first data on pre-diagnosis concentrations of sRANKL and OPG and risk of breast cancer-specific and overall mortality after a breast cancer diagnosis.. Two thousand six pre- and postmenopausal women with incident invasive breast cancer (1620 (81%) with ER+ disease) participating in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort were followed-up for mortality. Pre-diagnosis concentrations of sRANKL and OPG were quantified in baseline serum samples using an enzyme-linked immunosorbent assay and electrochemiluminescent assay, respectively. Hazard ratios (HRs) and 95% confidence intervals (CIs) for breast cancer-specific and overall mortality were calculated using Cox proportional hazards regression models.. High pre-diagnosis endogenous concentrations of OPG, the decoy receptor for RANKL, were associated with increased risk of death after a breast cancer diagnosis, especially in those with ER+ disease. These results need to be confirmed in well-characterized patient cohorts.

Topics: Adult; Aged; Breast Neoplasms; Case-Control Studies; Cohort Studies; Female; Humans; Limit of Detection; Middle Aged; Osteoprotegerin; Prognosis; RANK Ligand; Risk

The role of direct cell-cell interactions mediating selective bone metastasis by breast cancer cells (BCCs) niche is still mostly unknown.. Conditioned medium and direct cell-cell contacts experiments were used to investigate the effect of bone marrow-derived mesenchymal stromal cells (MSCs), osteoprogenitor-like cells (MG-63) and osteosarcoma cells (SaOS-2) on luminal-like (MCF-7) and basal-like (MDA-MB-231) BCCs flow cytometry was used to assess the purity of isolated BCCs and osteoblasts. Expression of osteoblastic markers was investigated by semi-quantitative RT-PCR. RANKL and OPG levels were measured by ELISA.. Conditioned medium from MSCs and osteoblasts induced the expression of osteoblastic markers in BCCs. While co-culture assays with SaOS-2 increased the expression of osteoblastic markers in MCF-7 cells, SaOS-2 cell conditioned medium increased the expression of RANKL, PTHrP, VEGF and NOGGIN in MCF-7 cells. Co-cultures with either MG-63 cells or MSCs induced OPG and MMP-2 in both tumor cell lines. Interestingly, conditioned medium from co-cultures of MSCs and MDA-MB-231 cells significantly decreased the proliferation of activated T lymphocytes which was reversed by addition of anti-OPG antibodies to the co-cultures.. Our data suggest that MSCs strongly contribute to the adaptation and invasiveness of breast cancer cells in skeletal tissues.

Topics: Bone Marrow Cells; Breast Neoplasms; Cell Line; Cell Line, Tumor; Cell Proliferation; Coculture Techniques; Female; Humans; Mesenchymal Stem Cells; Osteoblasts; Osteoprotegerin; RANK Ligand; T-Lymphocytes

Despite consistent improvements in diagnostic and therapeutic strategies for breast cancer, up to 40% of patients will develop bone metastases. To reduce the morbidity and complications related with bone metastases, it is imperative to reduce their etiological factors. Osteoporosis, being characterized by a sudden estrogen deficiency, may provide a favorable condition for bone metastasis. This work, using a humanized 3D in vitro model, aims at evaluating the relationship between osteoporosis and breast cancer-derived bone metastases. Bone tissue discarded from total hip replacement surgery of healthy and osteoporotic patients was cultured in a rolling apparatus system in hypoxic environment. Protein levels (i.e., vascular endothelial growth factor (VEGF), VEGF receptor 1, VEGF receptor 2, interleukin (IL)-6, IL-1β, IL-8 IL-10, tumor necrosis factor α (TNF-α), osteoprotegerin (OPG), receptor activator for nuclear factor KB ligand (RANKL)) and histological and immunohistochemical (i.e., cytokeratin 8 and 18) analyses showed a noticeable specificity of breast cancer cells for the colonization of osteoporotic bone. These data are the first to demonstrate that using humanized 3D in vitro systems, which individually model the pre- and postmenopausal bone microenvironment, it is possible to recognize major differences in tumor growth and colonization between healthy and osteoporotic status. Thus, this system might help to develop a shared system between basic and clinical sciences where a personalized diagnosis is associated to a therapeutic strategy designed for a single patient: a model able to achieve a translational research approach in the clinical setting, which may lead to the application and dissemination of personalized medicine. J. Cell. Physiol. 232: 1826-1834, 2017. © 2016 Wiley Periodicals, Inc.

Topics: Aged; Aged, 80 and over; Bone and Bones; Breast Neoplasms; Cell Line, Tumor; Cytokines; Enzyme-Linked Immunosorbent Assay; Female; Femur Head; Humans; Immunohistochemistry; Middle Aged; Models, Biological; Osteoporosis; Osteoprotegerin; RANK Ligand; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factor Receptor-2

Osteoprotegerin (OPG) is a robust antiresorptive molecule that acts as a decoy receptor for the receptor activator of nuclear factor κB ligand (RANKL), the mediator of osteoclastogenesis. This study was designed to explore the possible role of serum OPG and RANKL in detecting bone metastasis in breast cancer and its interaction with clinicopathologic parameters. Serum levels of RANKL and OPG were estimated in 44 metastatic and 36 nonmetastatic breast cancer patients using ELISA kits. Serum OPG levels were significantly reduced in patients with bone metastasis and correlated negatively with the number of bone lesions and CA 15-3 levels. At concentrations ≤82 pg/ml, OPG showed a high specificity in identifying the presence of bone metastasis (92%), albeit with low sensitivity (59%), which improved after the exclusion of diabetics and patients treated with aromatase inhibitors (AI). Serum RANKL levels were significantly higher in the presence of bone metastasis and hypercalcemia. At concentrations >12.5 pg/ml, RANKL had an associated sensitivity of 86%, albeit with low specificity (53%), in detecting bone metastasis. The RANKL/OPG ratio significantly increased in the presence of bone metastasis with appropriate sensitivity and specificity (73% and 72%, respectively) at a cutoff of ≥0.14 for the detection of bone metastasis. Serum OPG and RANKL/OPG ratios are promising biomarkers for detecting bone metastasis in breast cancer patients.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Bone Neoplasms; Breast Neoplasms; Female; Humans; Middle Aged; Neoplasm Grading; Osteoprotegerin; RANK Ligand; ROC Curve

Breast cancer is the most common female cancer, affecting approximately one in eight women during their lifetime in North America and Europe. Receptor Activator of NF-kB Ligand (RANKL), its receptor RANK and the natural antagonist osteoprotegerin (OPG) are essential regulators of bone resorption. We have initially shown that RANKL/RANK are essential for hormone-driven mammary epithelial proliferation in pregnancy and RANKL/RANK have been implicated in mammary stem cell biology. Using genetic mouse-models, we and others identified the RANKL/RANK system as a key regulator of sex hormone, BRCA1-mutation, and oncogene-driven breast cancer and we proposed that RANKL/RANK might be involved in the initiation of breast tumors. We now report that in postmenopausal women without known genetic predisposition, high RANKL and progesterone serum levels stratify a subpopulation of women at high risk of developing breast cancer 12-24 months before diagnosis (5.33-fold risk, 95%CI 1.5-25.4; P=0.02). In women with established breast cancer, we demonstrate that RANKL/OPG ratios change dependent on the presence of circulating tumor cells (CTCs). Finally, we show in a prospective human breast cancer cohort that alterations in RANKL/OPG ratios are significantly associated with breast cancer manifestation. These data indicate that the RANKL/RANK/OPG system is deregulated in post-menopausal women at high risk for breast cancer and in women with circulating tumor cells. Thus, serum levels of RANKL/OPG are potentially indicative of predisposition and progression of breast cancer in humans. Advancement of our findings towards clinical application awaits prior validation in independent patient cohorts.

Topics: Biomarkers, Tumor; Breast Neoplasms; Early Detection of Cancer; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplastic Cells, Circulating; Osteoprotegerin; Postmenopause; Progesterone; Prospective Studies; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Up-Regulation

It is widely recognized that inflammation promotes breast cancer invasion and metastasis. Given the complex nature of the breast tumor inflammatory microenvironment, much remains to be understood of the molecular mechanisms that govern these effects. We have previously shown that osteoprotegerin knockdown in breast cancer cells resulted in reduced invasion and metastasis. Here we present novel insight into the role of osteoprotegerin in inflammation-driven tumor progression in breast cancer by investigating the link between osteoprotegerin, macrophages and the potent pro-inflammatory cytokine Interleukin-1beta.. We used human breast cancer cell lines to investigate the effects of Interleukin-1beta treatment on osteoprotegerin secretion as measured by ELISA. We analyzed public datasets containing human breast cancer genome-wide mRNA expression data to reveal a significant and positive correlation between osteoprotegerin mRNA expression and the mRNA expression of Interleukin-1beta and of monocyte chemoattractant protein CC-chemokine ligand 2. Osteoprotegerin, Interleukin-1beta and CC-chemokine ligand 2 mRNA levels were also examined by qPCR on cDNA from normal and cancerous human breast tissue. We determined the effect of Interleukin-1beta-producing macrophages on osteoprotegerin expression by co-culturing breast cancer cells and differentiated THP-1 macrophages. Immunohistochemistry was performed on human breast tumor tissue microarrays to assess macrophage infiltration and osteoprotegerin expression. To demonstrate that osteoprotegerin mediated functional effects of Interleukin-1beta we performed cell invasion studies with control and OPG siRNA knockdown on Interleukin-1beta-treated breast cancer cells.. We report that Interleukin-1beta induces osteoprotegerin secretion, independent of breast cancer subtype and basal osteoprotegerin levels. Co-culture of breast cancer cells with Interleukin-1beta-secreting macrophages resulted in a similar increase in osteoprotegerin secretion in breast cancer cells as Interleukin-1beta treatment. Macrophage infiltration correlates with osteoprotegerin secretion in human breast tumor tissue samples. We show that osteoprotegerin secretion is regulated by Interleukin-1beta in a p38- and p42/44-dependent manner. We also demonstrate that osteoprotegerin knockdown represses Interleukin-1beta expression, Interleukin-1beta-mediated breast cancer cell invasion and MMP3 expression.. These data indicate a novel role for osteoprotegerin as a mediator of inflammation- promoted breast cancer progression.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Coculture Techniques; Computational Biology; Cytokines; Databases, Genetic; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Inflammation Mediators; Interleukin-1beta; Macrophages; MAP Kinase Signaling System; Osteoprotegerin; Tumor Microenvironment

Circulating osteoprotegerin (OPG), a member of the receptor activator of nuclear factor kappa-B (RANK) axis, may influence breast cancer risk via its role as the decoy receptor for both the RANK ligand (RANKL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Circulating OPG and breast cancer risk has been examined in only one prior study.. A case-control study was nested in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. A total of 2008 incident invasive breast cancer cases (estrogen receptor (ER)+, n = 1622; ER-, n = 386), matched 1:1 to controls, were included in the analysis. Women were predominantly postmenopausal at blood collection (77%); postmenopausal women included users and non-users of postmenopausal hormone therapy (HT). Serum OPG was quantified with an electrochemiluminescence assay. Relative risks (RRs) and 95% confidence intervals (CIs) were calculated using conditional logistic regression.. The associations between OPG and ER+ and ER- breast cancer differed significantly. Higher concentrations of OPG were associated with increased risk of ER- breast cancer (top vs. bottom tertile RR = 1.93 [95% CI 1.24-3.02]; p. This study provides the first prospective data on OPG and breast cancer risk by hormone receptor subtype. High circulating OPG may represent a novel risk factor for ER- breast cancer.

Topics: Breast Neoplasms; Case-Control Studies; Cohort Studies; Female; Humans; Middle Aged; Osteoprotegerin; Prospective Studies; Risk Factors

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

Aromatase inhibitor (AI) therapy is associated with musculoskeletal (MS) toxicity, which adversely affects quality of life and therapy adherence. Our objective was to evaluate whether genetic variants may predict endocrine therapy-related MS pain and hot flashes in a prospective observational cohort study.. 254 early breast cancer patients starting AI (n = 159) or tamoxifen therapy (n = 95) were included in this genetic biomarker study. MS and vasomotor symptoms were assessed at baseline and after 3, 6 and 12 months of therapy. AI-induced MS pain was defined as an increase in arthralgia or myalgia relative to baseline. Single nucleotide polymorphisms (SNP) in candidate genes involved in oestrogen signalling or previously associated with AI-related MS pain or oestrogen levels were selected.. Overall, 13 SNPs in CYP19, CYP17, osteoprotegerin (OPG) and oestrogen receptor 1 exhibited an allele frequency >0.05 and were included in the analysis. Patients carrying the G allele of rs2073618 in OPG experienced significantly more AI-induced MS toxicity compared to the wildtype allele, after correction for multiple testing (P = 0.046). Furthermore, this SNP was associated with severity of pain (P = 0.018). No association was found with regard to the other SNPs, both in AI and tamoxifen-treated patients. Neither could an association with vasomotor symptoms be demonstrated.. The SNP rs2073618 in OPG is associated with an increased risk of MS symptoms and pain with AI therapy, which has not been reported previously. Validation of this finding in larger cohorts and further functional studies are required.

Topics: Aged; Aged, 80 and over; Antineoplastic Agents, Hormonal; Aromatase Inhibitors; Arthralgia; Breast Neoplasms; Female; Genetic Association Studies; Genetic Predisposition to Disease; Humans; Longitudinal Studies; Middle Aged; Musculoskeletal Pain; Myalgia; Osteoprotegerin; Pain Measurement; Phenotype; Polymorphism, Single Nucleotide; Prospective Studies; Risk Factors; Time Factors

The crosstalk between malignant and nonmalignant cells in the tumor microenvironment, as maneuvered by cytokines/chemokines, drives breast cancer progression. In our previous study, we discovered Osteoprotegerin (OPG) as one of the cytokines heavily secreted by breast cancer cells. We demonstrated that OPG is expressed and secreted at very high levels from the highly invasive breast cancer cell lines SUM149PT and SUM1315MO2 as compared to normal human mammary epithelial HMEC cells. OPG was involved in modulating aneuploidy, cell proliferation, and angiogenesis in breast cancer. Mass spectrometry analysis performed in this study revealed OPG interacts with fatty acid synthase (FASN), which is a key enzyme of the fatty acid biosynthetic pathway in breast cancer cells. Further, electron microscopy, immunofluorescence, and fluorescence quantitation assays highlighted the presence of a large number of lipid bodies (lipid droplets) in SUM149PT and SUM1315MO2 cells in comparison to HMEC. We recently showed upregulation of the COX-2 inflammatory pathway and its metabolite PGE2 secretion in SUM149PT and SUM1315MO2 breast cancer cells. Interestingly, human breast cancer tissue samples displayed high expression of OPG, PGE2 and fatty acid synthase (FASN). FASN is a multifunctional enzyme involved in lipid biosynthesis. Immunofluorescence staining revealed the co-existence of COX-2 and FASN in the lipid bodies of breast cancer cells. We reasoned that there might be crosstalk between OPG, FASN, and COX-2 that sustains the inflammatory pathways in breast cancer. Interestingly, knocking down OPG by CRISPR/Cas9 gene editing in breast cancer cells decreased FASN expression at the protein level. Here, we identified cis-acting elements involved in the transcriptional regulation of COX-2 and FASN by recombinant human OPG (rhOPG). Treatment with FASN inhibitor C75 and COX-2 inhibitor celecoxib individually decreased the number of lipid bodies/cell, downregulated phosphorylation of ERK, GSK3β, and induced apoptosis by caspase-3/7 and caspase-9 activation. But a more efficient and effective decrease in lipid bodies/cell and survival kinase signaling was observed upon combining the drug treatments for the aggressive cancer cells. Collectively, the novel biological crosstalk between OPG, FASN, and COX-2 advocates for combinatorial drug treatment to block these players of carcinogenesis as a promising therapeutic target to treat highly invasive breast cancer.

Topics: 4-Butyrolactone; Breast Neoplasms; Carcinogenesis; Caspase 9; Celecoxib; Cell Line, Tumor; Cyclooxygenase 2 Inhibitors; Fatty Acid Synthases; Female; Humans; Lipid Droplets; Lipid Metabolism; Molecular Targeted Therapy; Osteoprotegerin; Protein Binding; Receptor Cross-Talk; Signal Transduction; Tumor Microenvironment

Objective To construct a recombinant adenovirus expressing siRNA targeting human sclerostin (SOST) gene, and test the function of MG-63 cells while co-cultured with MDA-MB-231 cells infected by Ad-siSOST. MethodsAccording to the RNA sequence of SOST gene, two pairs of primers which contained 3 siRNA sequences were designed, and a pB2B plasmid was taken as template to amplify 2 DNA sequences. Both of the 2 DNA sequences were ligated to pAdTrace-OK by Gibson DNA Assembly way. After homologous recombination between recombinant shuttle plasmid and adenovirus vector plasmid, the adenovirus was packaged in HEK-293 cells. PCR and ELISA were used to test the expression of SOST in MDA-MB-231 cells which were infected with Ad-siSOST. In the co-culture system of MG-63 cells and MDA-MB-231 cells infected Ad-siSOST, osteoprotegerin (OPG), osteocalcin (OCN), integrin binding sialoprotein (IBSP) and receptor activator of nuclear factor-κB ligand (RANKL) were tested by quantitative real-time PCR. Results Recombinant shuttle plasmid which contained 3 interfering fragments was constructed successfully, and Ad-siSOST was obtained after homologous recombination and packaging. SOST expression in MDA-MB-231 cells was downregulated significantly after infeceted with Ad-siSOST. The mRNA levels of OPG, OCN, IBSP in MG-63 cells increased significantly, while the level of RANKL mRNA decreased significantly, and all 4 gene expressions were reversed after the infection of Ad-siSOST. Conclusion Knockdown of SOST in MG-63 cells increases osteogenesis and ratio of OPG/RANKL in vitro.

Topics: Adaptor Proteins, Signal Transducing; Bone Morphogenetic Proteins; Breast Neoplasms; Cell Line, Tumor; Coculture Techniques; Genetic Markers; HEK293 Cells; Humans; Integrin-Binding Sialoprotein; Osteoblasts; Osteocalcin; Osteogenesis; Osteoprotegerin; RANK Ligand; Real-Time Polymerase Chain Reaction

Emerging evidence suggests a role of receptor activator of nuclear factor κB (RANK)/RANK ligand (RANKL) signaling in breast cancer development. Lower osteoprotegerin (OPG) levels, the endogenous decoy receptor for RANKL which competes with RANK for binding of RANKL, has been reported among BRCA mutation carriers. Whether low OPG levels contribute to the high breast cancer risk in this population is unknown. OPG concentrations were measured in plasma of 206 cancer-free BRCA mutation carriers using an enzyme-linked immunosorbent assay. Subjects were categorized as high vs. low based on the median of the entire cohort (95 ng/mL) and followed for a new diagnosis of breast cancer. Cumulative incidence by baseline plasma OPG concentration was estimated using Kaplan-Meier survival analysis. Cox proportional hazards models were used to estimate the adjusted hazard ratios for the association between plasma OPG and breast cancer risk. Over a mean follow-up period of 6.5 years (range 0.1-18.8 years), 18 incident breast cancer cases were observed. After ten years of follow-up, the cumulative incidence of breast cancer among women with low OPG was 21%, compared to 9% among women with high OPG (P-log rank = 0.046). After multivariate adjustment, women with high plasma OPG had a significantly decreased risk of developing breast cancer, compared to women with low OPG (HR = 0.25; 95%CI 0.08-0.78; P = 0.02). These data suggest that low OPG levels are associated with an increased risk of BRCA-associated breast cancer. Targeting RANK signalling may represent a plausible, non-surgical prevention option for BRCA mutation carriers.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; BRCA1 Protein; BRCA2 Protein; Breast Neoplasms; Female; Heterozygote; Humans; Kaplan-Meier Estimate; Middle Aged; Mutation; Osteoprotegerin; Proportional Hazards Models; Risk Assessment; Risk Factors; Young Adult

Chemotherapy induced ovarian failure (CIOF) results in rapid bone loss. Receptor Activator of Nuclear Factor Kappa-B (RANK)-RANK ligand (RANK-L) signaling balances bone resorption and formation. Osteoprotegerin (OPG) acts as a decoy receptor for RANK, interrupting osteoclast activation and bone resorption. This study examined the relationship between OPG and bone loss in women with CIOF.. Premenopausal women with stage I/II breast cancers receiving adjuvant chemotherapy were evaluated at chemotherapy initiation, 6 and 12 months. Bone mineral density (BMD) at the lumbar spine (LS) and femoral neck (FN), follicle stimulating hormone (FSH), ionized calcium, osteocalcin, and OPG were serially measured. CIOF was defined as a negative pregnancy test, FSH levels >30 MIU/mL, and ≥3 months of amenorrhea.. Forty women were enrolled; 31 (77.5%) met CIOF criteria. BMD significantly decreased (p < 0.001) in the CIOF group at both time points: LS BMD decreased from a median of 0.993 g/cm(2) to 0.976 g/cm(2) and 0.937 g/cm(2) at 6 and 12 months, respectively. OPG was significantly elevated at 6 months (median increase 0.30 pmol/L, p = 0.015) and then decreased at 12 months to levels still above baseline (median difference 0.2 pmol/L, p = 0.70).. In what was likely a compensatory response to rapid bone loss, CIOF patients' OPG levels increased at 6 months and then decreased at 12 months to values greater than baseline assessments. This phenomenon is described in other diseases, but never before in CIOF.

Topics: Adult; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Chemotherapy, Adjuvant; Cyclophosphamide; Doxorubicin; Female; Fluorouracil; Humans; Methotrexate; Middle Aged; Osteoporosis; Osteoprotegerin; Primary Ovarian Insufficiency

Dormant disseminated tumour cells can be detected in the bone marrow of breast cancer patients several years after resection of the primary tumour. The majority of these patients will remain asymptomatic, however, ∼ 15% will go on to develop overt bone metastases and this condition is currently incurable. The reason why these dormant cells are stimulated to proliferate and form bone tumours in some patients and not others remains to be elucidated. We have recently shown that in an in vivo model, increasing bone turnover by ovariectomy stimulated proliferation of disseminated tumour cells, resulting in formation of bone metastasis. We now show for the first time that osteoclast mediated mechanisms induce growth of tumours from dormant MDA-MB-231 cells disseminated in the bone. We also show that disruption of RANK-RANKL interactions following administration of OPG-Fc inhibits growth of these dormant tumour cells in vivo. Our data support early intervention with anti-resorptive therapy in a low-oestrogen environment to prevent development of bone metastases.

Topics: Animals; Bone Marrow Cells; Bone Neoplasms; Bone Resorption; Breast Neoplasms; Cell Proliferation; Disease Models, Animal; Female; Humans; Immunoglobulin Fc Fragments; Mice; Osteoclasts; Osteoprotegerin; Ovariectomy; RANK Ligand

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 is the major cause of cancer death in women worldwide. The most common site of metastasis is bone. Bone metastases obstruct the normal bone remodeling process and aberrantly enhance osteoclast-mediated bone resorption, which results in osteolytic lesions. 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is an endogenous ligand of peroxisome proliferator-activated receptor gamma (PPARγ) that has anti-inflammatory and antitumor activity at micromolar concentrations through PPARγ-dependent and/or PPARγ-independent pathways. We investigated the inhibitory activity of 15d-PGJ2 on the bone loss that is associated with breast cancer bone metastasis and estrogen deficiency caused by cancer treatment. 15d-PGJ2 dose-dependently inhibited viability, migration, invasion, and parathyroid hormone-related protein (PTHrP) production in MDA-MB-231 breast cancer cells. 15d-PGJ2 suppressed receptor activator of nuclear factor kappa-B ligand (RANKL) mRNA levels and normalized osteoprotegerin (OPG) mRNA levels in hFOB1.19 osteoblastic cells treated with culture medium from MDA-MB-231 cells or PTHrP, which decreased the RANKL/OPG ratio. 15d-PGJ2 blocked RANKL-induced osteoclastogenesis and inhibited the formation of resorption pits by decreasing the activities of cathepsin K and matrix metalloproteinases, which are secreted by mature osteoclasts. 15d-PGJ2 exerted its effects on breast cancer and bone cells via PPARγ-independent pathways. In Balb/c nu/nu mice that received an intracardiac injection of MDA-MB-231 cells, subcutaneously injected 15d-PGJ2 substantially decreased metastatic progression, cancer cell-mediated bone destruction in femora, tibiae, and mandibles, and serum PTHrP levels. 15d-PGJ2 prevented the destruction of femoral trabecular structures in estrogen-deprived ICR mice as measured by bone morphometric parameters and serum biochemical data. Therefore, 15d-PGJ2 may be beneficial for the prevention and treatment of breast cancer-associated bone diseases.

Topics: Anilides; Animals; Bone Neoplasms; Bone Resorption; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Survival; Disease Models, Animal; Estrogens; Female; Humans; Male; Mice; Mice, Nude; Osteoclasts; Osteolysis; Osteoprotegerin; Ovariectomy; Parathyroid Hormone-Related Protein; PPAR gamma; Prostaglandin D2; RANK Ligand

To develop new and effective chemopreventive agents against bone metastasis, we assessed the effects of muscadine grape skin extract (MSKE), whose main bioactive component is anthocyanin, on bone turnover, using prostate and breast cancer cell models overexpressing Snail transcription factor. MSKE has been shown previously to promote apoptosis in prostate cancer cells without affecting normal prostate epithelial cells. Snail is overexpressed in prostate and breast cancer, and is associated with increased invasion, migration and bone turnover/osteoclastogenesis. Cathepsin L (CatL) is a cysteine cathepsin protease that is overexpressed in cancer and involved in bone turnover. Snail overexpression in prostate (LNCaP, ARCaP-E) and breast (MCF-7) cancer cells led to increased CatL expression/activity and phosphorylated STAT-3 (pSTAT-3), compared to Neo vector controls, while the reverse was observed in C4-2 (the aggressive subline of LNCaP) cells with Snail knockdown. Moreover, CatL expression was higher in prostate and breast tumor tissue compared to normal tissue. MSKE decreased Snail and pSTAT3 expression, and abrogated Snail-mediated CatL activity, migration and invasion. Additionally, Snail overexpression promoted osteoclastogenesis, which was significantly inhibited by the MSKE as effectively as Z-FY-CHO, a CatL-specific inhibitor, or osteoprotegerin, a receptor activator of nuclear factor kappa B ligand (RANKL) antagonist. Overall, these novel findings suggest that Snail regulation of CatL may occur via STAT-3 signaling and can be antagonized by MSKE, leading to decreased cell invasion, migration and bone turnover. Therefore, inhibition using a natural product such as MSKE could potentially be a promising bioactive compound for bone metastatic cancer.

Topics: Animals; Anticarcinogenic Agents; Apoptosis; Bone Neoplasms; Breast Neoplasms; Cathepsin L; Cell Line, Tumor; Cell Movement; Chemoprevention; Female; Humans; Male; MCF-7 Cells; Mice; Mice, Nude; Neoplasm Invasiveness; Osteoclasts; Osteogenesis; Osteoprotegerin; Plant Extracts; Prostatic Neoplasms; RANK Ligand; Signal Transduction; Snail Family Transcription Factors; STAT3 Transcription Factor; Transcription Factors; Vitis

Breast cancer patients treated with aromatase inhibitors (AIs) may experience musculoskeletal adverse events (MS-AEs). Several studies have confirmed that the RANKL/RANK/OPG signaling pathway plays a dominant role in bone health. Therefore, this study aimed to analyze the relationship between the serum levels of RANKL, OPG and their SNPs (single nucleotide polymorphisms) with AI-related MS-AEs.. Patients with early stage, hormone-sensitive breast cancer who were receiving AI therapy were enrolled. We included 208 cases with AI-related MS-AEs and 212 without (controls). The levels of estradiol, bone-turnover markers, multiple inflammatory cytokines, RANKL,OPG and lumbar spine BMD were measured, and questionnaires were completed. We analyzed 29 SNPs of RANKL, RANK and OPG using Sequenom MassARRAY assays and PCR-based TaqMan assays. The levels of bone-turnover markers and RANKL and the ratio of RANKL/OPG were higher in patients with AI-related MS-AEs than controls (all p < 0.05). A genetic assay showed that the RANKL SNP rs7984870 and OPG SNP rs2073618 were associated with AI-related MS-AEs. In patients with AI-related MS-AEs, rs7984870 CC and rs2073618 CC were risk genotypes. Carriers of the rs7984870 CC genotype were more likely to have a higher RANKL level and RANKL/OPG ratio than carriers of the GG genotype, and carriers of the rs2073618 CC genotype were more likely to have a lower OPG level and a higher RANKL/OPG ratio than carriers of the GG genotype (all p < 0.05). Moreover, risk genotypes were associated with higher levels of serum CTX and PINP and a lower lumbar spine BMD (all p < 0.05).. In conclusion, the RANKL and OPG risk genotypes synergize to negatively impact bone health and predispose breast cancer patients to AI-related MS-AEs.

Topics: Aromatase Inhibitors; Asian People; Breast Neoplasms; Case-Control Studies; Female; Follow-Up Studies; Genetic Predisposition to Disease; Genotype; Humans; Middle Aged; Musculoskeletal Diseases; Neoplasm Grading; Osteoprotegerin; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Prognosis; RANK Ligand

Osteoprotegerin (OPG) is a glycoprotein that has multifaceted role and is associated with several cancer malignancies like that of bladder carcinoma, gastric carcinoma, prostate cancer, multiple myeloma and breast cancer. Also OPG has been associated with several organ pathologies. The widespread expression of OPG suggests that OPG may have multiple biological activities that are yet to be explored.. The anchorage-independent sphere cultures of the adherent cells were instrumental in our study as it provided a deeper insight into the complexity of a 3D tumor. Cytokine profiling was performed for OPG's detection in the microenvironment. ELISA and western blotting were performed to quantify the OPG secretion and measure the protein levels respectively. OPG expression was detected in human breast cancer tissue samples by IHC. To decipher OPG's role in tumor aggressiveness both recombinant human OPG as well as OPG rich and depleted breast cancer cell conditioned media were tested. Western blotting and MTT assay were performed to detect changes in signaling pathways and proliferation that were induced in presence of OPG. Onset of aneuploidy, in presence of OPG, was measured by cell cycle analysis and western blotting. Finally, human Breast Cancer qBiomarker Copy Number PCR Array was used to detect how OPG remarkably induced gene copy numbers for oncogenic pathway regulators.. SUM149PT and SUM1315M02 cells secrete high levels of the cytokine OPG compared to primary human mammary epithelial cells (HMEC). High expression of OPG was also detected in human breast cancer tissue samples compared to the uninvolved tissue from the same patient. OPG induced proliferation of control HMEC spheres and triggered the onset of aneuploidy in HMEC sphere cultures. OPG induced the expression of aneuploidy related kinases Aurora-A Kinase (IAK-1), Bub1 and BubR1 probably through the receptor activator of nuclear factor kappa-B ligand (RANKL) and syndecan-1 receptors via the Erk, AKT and GSK3(3 signaling pathway. Gene copy numbers for oncogenic pathway regulators such AKT1, Aurora-A Kinase (AURKA or IAK-1), epidermal growth factor receptor (EGFR) and MYC with a reduction in the copy numbers of cyclin dependent kinase inhibitor 2A (CDKN2A), PTEN and DNA topoisomerase 2 alpha (TOP2A) were induced in presence of OPG.. These results highlight the role of OPG in reprogramming normal mammary epithelial cells to a tumorigenic state and suggest promising avenues for treating inflammatory breast cancer as well as highly invasive breast cancer with new therapeutic targets.

Topics: Aneuploidy; Breast Neoplasms; Cell Culture Techniques; Cell Line, Tumor; Cell Proliferation; Cytokines; Female; Gene Amplification; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Invasiveness; Osteoprotegerin; Tumor Microenvironment

Topics: Breast Neoplasms; Genes, BRCA1; Genes, BRCA2; Humans; Mutation; Osteoprotegerin

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

Many breast cancer patients experience bone metastases and suffer skeletal complications. The present study provides evidence on the protective and therapeutic potential of betulinic acid on cancer-associated bone diseases. Betulinic acid is a naturally occurring triterpenoid with the beneficial activity to limit the progression and severity of cancer, diabetes, cardiovascular diseases, atherosclerosis, and obesity. We first investigated its effect on breast cancer cells, osteoblastic cells, and osteoclasts in the vicious cycle of osteolytic bone metastasis. Betulinic acid reduced cell viability and the production of parathyroid hormone-related protein (PTHrP), a major osteolytic factor, in MDA-MB-231 human metastatic breast cancer cells stimulated with or without tumor growth factor-β. Betulinic acid blocked an increase in the receptor activator of nuclear factor-kappa B ligand (RANKL)/osteoprotegerin ratio by downregulating RANKL protein expression in PTHrP-treated human osteoblastic cells. In addition, betulinic acid inhibited RANKL-induced osteoclastogenesis in murine bone marrow macrophages and decreased the production of resorbed area in plates with a bone biomimetic synthetic surface by suppressing the secretion of matrix metalloproteinase (MMP)-2, MMP-9, and cathepsin K in RANKL-induced osteoclasts. Furthermore, oral administration of betulinic acid inhibited bone loss in mice intra-tibially inoculated with breast cancer cells and in ovariectomized mice causing estrogen deprivation, as supported by the restored bone morphometric parameters and serum bone turnover markers. Taken together, these findings suggest that betulinic acid may have the potential to prevent bone loss in patients with bone metastases and cancer treatment-induced estrogen deficiency.

Topics: Administration, Oral; Animals; Betulinic Acid; Bone Neoplasms; Bone Resorption; Breast Neoplasms; Cathepsin K; Cell Differentiation; Cell Line, Tumor; Cell Survival; Down-Regulation; Estrogens; Female; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Inbred ICR; Osteoclasts; Osteoprotegerin; Parathyroid Hormone-Related Protein; Pentacyclic Triterpenes; RANK Ligand; Triterpenes

Recently, it was found that α-Calcitonin gene-related peptide (CGRP) was associated with breast cancer metastases, but the role of CGRP in interaction between breast cancer and osteoblast during bone metastases is not clear. Here, we investigated the effect of CGRP on osteoblast in co-culture system with breast cancer. Using a breast cancer-osteoblast co-culture system, we chose MDA-MB-231 for breast cancer and human cell line MG-63 for osteoblast. CGRP was added to this co-culture system. The expression levels of the Runx2, RANK1, and osteoprotegerin (OPG) were analyzed using real-time PCR and western blot. CGRP receptors were investigated by immunofluorescence. We found that breast cancer cells cause osteolysis lesions by upregulating Runx2 expression, decreasing OPG expression, and increasing RANKL expression in osteoblasts. Our data prove that CGRP can regulate osteoclast coupling genes in osteoblast by increasing OPG, and decreasing RANKL and Runx2 expressions in a time-dependent manner; and inhibit those osteolytic factors induced by interaction between breast cancer cells and osteoblast. This inhibition could be abolished by the CGRP antagonist, CGRP8-37. In conclusion, calcitonin receptor-like receptor is the key player for CGRP's effect in this co-culture system.

Topics: Bone Neoplasms; Breast Neoplasms; Calcitonin Gene-Related Peptide; Cell Line, Tumor; Coculture Techniques; Gene Expression Regulation; Humans; Osteoblasts; Osteolysis; Osteoprotegerin; Peptide Fragments; RANK Ligand

Osteoprotegerin (OPG) is a secreted member of the TNF receptor superfamily, which binds to the receptor activator of nuclear factor κB ligand (RANKL) and inhibits osteoclast activity and bone resorption. Systemic administration of recombinant OPG was previously shown to inhibit tumor growth in bone and to prevent cancer-induced osteolysis. In this study, we examined the effect of OPG, when produced locally by breast cancer cells located within bone, using a mouse model of osteolytic breast cancer. MDA-MB-231-TXSA breast cancer cells, tagged with a luciferase reporter gene construct and engineered to overexpress full-length human OPG, were transplanted directly into the tibial marrow cavity of nude mice. Overexpression of OPG by breast cancer cells protected the bone from breast cancer-induced osteolysis and diminished intra-osseous tumor growth but had no effect on extra-skeletal tumor growth. This effect was associated with a significant reduction in the number of osteoclasts that lined the bone surface, resulting in a net increase in bone volume. Despite limiting breast cancer-mediated bone loss, OPG overexpression resulted in a significant increase in the incidence of pulmonary metastasis. Our results demonstrate that inhibition of osteoclastic bone resorption by OPG when secreted locally by tumors in bone may affect the behaviour of cancer cells within the bone microenvironment and their likelihood of spreading and establishing metastasis elsewhere in the body.

Topics: Animals; Bone Neoplasms; Bone Resorption; Breast Neoplasms; Cell Line, Tumor; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Humans; Mice; Mice, Nude; Neoplasm Invasiveness; Osteolysis; Osteoprotegerin; Soft Tissue Neoplasms; Transfection; X-Ray Microtomography

While their negative impact on bone health is well established, the effects of aromatase inhibition (AI) on Wnt inhibitors and osteoprotegerin (OPG) are unknown. The aim of the study was to investigate the effects of AI on serum levels of sclerostin, DKK-1 and OPG, as well as their associations with PINP and CTX as markers of bone turnover and bone mineral density (BMD) assessed by DXA.. We conducted a prospective longitudinal analysis of 70 postmenopausal women with hormone receptor-positive early breast cancer (BC) treated with anastrozole. All measurements were performed at baseline, 12 and 24 months of treatment. We measured the association of the investigated variables with circulating bone turnover markers, as well as with the BMD.. After 24 months of AI therapy, sclerostin and OPG concentrations increased from 29.5 pmol/l (SD = 15.1) and 6.8 pmol/l (SD = 2.2) at baseline to 43.2 pmol/l (SD = 20.6) (p < 0.001) and 7.4 pmol/l (SD = 2.2) (p = 0.028), respectively. DKK-1 levels decreased from 34.3 pmol/l (SD = 13.5) at baseline to 29.7 pmol/l (SD = 12.3) at the 24-month visit (p = 0.005). Sclerostin levels significantly correlated with PTH, OPG and BMD of the lumbar spine, while DKK-1 correlated with the BMD of the femoral neck and of the total hip.. The observed increase in sclerostin levels indicates a central role of osteocytes in bone turnover in women with BC.

Topics: Adaptor Proteins, Signal Transducing; Aged; Antineoplastic Agents; Aromatase Inhibitors; Biomarkers, Tumor; Bone Density; Bone Morphogenetic Proteins; Bone Neoplasms; Bone Remodeling; Breast Neoplasms; Enzyme-Linked Immunosorbent Assay; Female; Follow-Up Studies; Genetic Markers; Humans; Intercellular Signaling Peptides and Proteins; Middle Aged; Osteocytes; Osteoprotegerin; Postmenopause; Prospective Studies; Time Factors; Wnt Proteins

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

Bone metastases arise in nearly 70% of patients with advanced breast cancer, but the complex metastatic process has not been completely clarified yet. RANKL/RANK/OPG pathway modifications and the crosstalk between metastatic cells and bone have been indicated as potential drivers of the process. Interactions between tumor and bone cells have been studied in vivo and in vitro, but specific effects of the direct contact between human metastatic cells and human bone cells on RANKL/RANK/OPG pathway have not been investigated.. We directly co-cultured bone metastatic human breast cancer cells (BOKL) with osteo-differentiated human mesenchymal cells (BMSCs) from 3 different donors. BMSCs and BOKL were then enzymatically separated and FACS sorted. We found a significant increase in the RANKL/OPG ratio as compared to control, which was not observed in BOKL cultured in medium conditioned by BMSCs, neither in BOKL directly cultured with fibroblasts or medium conditioned by fibroblasts. Direct co-culture with osteo-differentiated BMSCs caused BOKL aggregation while proliferation was not affected by co-culture. To more specifically associate RANKL expression to osteogenic differentiation degree of BMSCs, we determined their osteogenic markers expression and matrix calcification relative to osteoblasts and fibroblasts.. In conclusion, our co-culture model allowed to demonstrate for the first time that direct contact but not paracrine interactions between human metastatic breast cancer cells and bone cells has a significant effect on RANKL/OPG expression in bone metastatic cells. Furthermore, only direct contact with the bone microenvironment induced BOKL clustering without however significantly influencing their proliferation and migration.

Topics: Bone Neoplasms; Breast Neoplasms; Cell Aggregation; Cell Communication; Cell Differentiation; Cell Movement; Cell Proliferation; Cellular Microenvironment; Coculture Techniques; Female; Gene Expression Regulation; Humans; Mesenchymal Stem Cells; Osteogenesis; Osteoprotegerin; RANK Ligand

The receptor activator of nuclear factor-kappaB ligand (RANKL), its signaling receptor RANK, and its natural decoy receptor OPG are members of the tumour necrosis factor (TNF) and TNF receptor superfamily and are best known for their essential role in controlling osteoclastogenesis. RANKL in bone has also been shown to serve as a chemoattractant for cancer cells, thu explaining the tropism of certain cancers such as breast and prostate cancer to preferentially metastasize to bone. Here, we will discuss the important role of RANKL and its possible role in the management of bone loss in patients with breast cancer.

Topics: Bone Resorption; Breast Neoplasms; Drug Design; Female; Humans; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B

The intravenous injection of bisphosphonates, currently used as treatment for osteoporosis, bone Paget's disease, multiple myeloma, or bone metastases, can cause jaw bone necrosis especially in consequence of trauma. The present research aimed to clarify the mechanisms underlying bone necrosis, exploring involvement of the oral mucosa "in vivo.". Specimens of oral mucosa were removed from bisphosphonate-treated patients with or without jaw bone necrosis. In mucosa specimens, expression was evaluated of: cytokines involved in the inflammatory process, factors involved in osteoclast activity, i.e., receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin, a factor involved in cell proliferation, namely hydroxymethylglutaryl coenzyme A reductase, and a factor involved in angiogenesis, namely vascular endothelial growth factor (VEGF).. Interleukin (IL)-6 and the RANK/osteoprotegerin ratio were significantly elevated in mucosa from patients with versus without jaw necrosis, whereas hydroxymethylglutaryl coenzyme A reductase and VEGF were significantly decreased.. Our results suggest that mucosa, stimulated by bisphosphonate released from the bone, can contribute to the development of jaw necrosis, reducing VEGF, and producing IL-6 in consequence of hydroxymethylglutaryl coenzyme A reductase reduction. In turn, IL-6 stimulates osteoclast activity, as shown by the increased RANKL/osteoprotegerin ratio.. The results of this study suggest the importance of evaluating during bisphosphonate treatment the production of IL-6, RANKL, osteoprotegerin, and VEGF, in order to monitor the jaw osteonecrosis onset. To avoid repeated mucosa excisions, the determination of these factors could be carried out in crevicular fluid.

Topics: Aged; Bisphosphonate-Associated Osteonecrosis of the Jaw; Bone Density Conservation Agents; Bone Neoplasms; Breast Neoplasms; Case-Control Studies; Cell Proliferation; Cytokines; Diphosphonates; Endothelial Cells; Female; Gingival Crevicular Fluid; Humans; Hydroxymethylglutaryl CoA Reductases; Imidazoles; Injections, Intravenous; Interleukin-6; Male; Middle Aged; Mouth Mucosa; Multiple Myeloma; Osteoclasts; Osteoprotegerin; RANK Ligand; Statistics, Nonparametric; Vascular Endothelial Growth Factor A; Zoledronic Acid

Breast cancer, the most common cancer affecting women in the USA and UK, is known to have a high frequency of osteolytic bone metastasis. Receptor activator of nuclear-κB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG) are a group of important regulators for osteoclast differentiation and activation. These molecules have been implicated in bone metastasis. Since the discovery of the triad of RANK, RANKL and OPG in healthy bone turnover, a better understanding of these factors in bone metastasis has been sought.. Using our clinical breast cancer cohort, the transcript levels of RANK, RANKL and OPG were examined using real-time quantitative-polymerase chain reaction (qPCR). Expression of these molecules in the immortalised breast cancer cell lines MCF-7 and MDA-MB-231 was also analyzed using qPCR following treatment with β-oestradiol in a concentration-dependent manner.. RANK, RANKL and OPG were all shown to be expressed in the breast cancer cell lines examined. Transcript levels were shown to be reduced in tumour samples when compared with normal tissue. Reduced RANK expression was associated with a worse clinical outcome and levels were significantly reduced in patients with general metastasis, bone metastasis and those who had died of the disease. Patients with reduced RANKL expression were more likely to develop local recurrence, bone metastasis or die from the disease. Using Kaplan-Meier survival analysis, lower expression levels of OPG were found to be associated with significantly better overall patient survival in our cohort.. The corresponding prognostic and therapeutic potential is yet to be further investigated. Our data suggest that RANK, RANKL and OPG may potentially be used as novel prognostic markers for bone metastasis and provide new therapeutic targets in the treatment of breast cancer.

Topics: Bone Neoplasms; Breast Neoplasms; Estrogens; Female; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; MCF-7 Cells; Molecular Targeted Therapy; Neoplasm Staging; Neoplasms, Hormone-Dependent; Osteoprotegerin; Prognosis; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B

The receptor activator of NF-κB (RANK), its ligand (RANKL) and osteoprotegerin (OPG) have been reported to play a role in the pathophysiological bone turnover and in the pathogenesis of breast cancer. Based on this we investigated the role of single nucleotide polymorphisms (SNPs) within RANK, RANKL and OPG and their possible association to breast cancer risk.. Genomic DNA was obtained from Caucasian participants consisting of 307 female breast cancer patients and 396 gender-matched healthy controls. We studied seven SNPs in the genes of OPG (rs3102735, rs2073618), RANK (rs1805034, rs35211496) and RANKL (rs9533156, rs2277438, rs1054016) using TaqMan genotyping assays. Statistical analyses were performed using the χ2-tests for 2 x 2 and 2 x 3 tables.. The allelic frequencies (OR: 1.508 CI: 1.127-2.018, p=0.006) and the genotype distribution (p=0.019) of the OPG SNP rs3102735 differed significantly between breast cancer patients and healthy controls. The minor allele C and the corresponding homo- and heterozygous genotypes are more common in breast cancer patients (minor allele C: 18.4% vs. 13.0%; genotype CC: 3.3% vs. 1.3%; genotype CT: 30.3% vs. 23.5%). No significantly changed risk was detected in the other investigated SNPs. Additional analysis showed significant differences when comparing patients with invasive vs. non-invasive tumors (OPG rs2073618) as well as in terms of tumor localization (RANK rs35211496) and body mass index (RANKL rs9533156 and rs1054016).. This is the first study reporting a significant association of the SNP rs3102735 (OPG) with the susceptibility to develop breast cancer in the Caucasian population.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Breast Neoplasms; Case-Control Studies; Chi-Square Distribution; Female; Gene Frequency; Genetic Predisposition to Disease; Germany; Humans; Middle Aged; Odds Ratio; Osteoprotegerin; Phenotype; Polymorphism, Single Nucleotide; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Risk Assessment; Risk Factors; White People; Young Adult

Receptor activator of nuclear factor-kappa B ligand (RANKL) is a TNF ligand superfamily member that is essential for the formation, activation, and function of osteoclasts. RANKL functions via its cognate receptor RANK, and it is inhibited by the soluble decoy receptor osteoprotegerin (OPG). In skeletal metastases, the ratio of RANKL to OPG is upregulated, which leads to increased osteoclast-mediated bone destruction. These changes in the bone microenvironment not only compromise the structural integrity of bone, leading to severe clinical morbidities, but have also been implicated in establishment of de novo bone metastasis and the progression of existing skeletal tumors. Evaluation of RANKL inhibitors, including the fully human anti-RANKL antibody denosumab, in patients with cancer has shown reductions in tumor-induced bone resorption activity and successful management of skeletal complications of bone metastases. RANKL also functions as a major paracrine effector of the mitogenic action of progesterone in mouse mammary epithelium, and it has a role in ovarian hormone-dependent expansion and regenerative potential of mammary stem cells. RANKL inhibition attenuates mammary tumorigenesis and pulmonary metastases in mouse models. These data suggest that the contribution of progesterone to increased mammary cancer incidence is mediated, at least in part, by RANKL-dependent changes in the mammary epithelium; RANKL also directly promotes distant metastases. In summary, the antitumor and antimetastatic effects of RANKL inhibition can occur by at least 2 distinct mechanisms, one in the bone via osteoclast-dependent effects, and the second via direct effects on the tumor cells of various origins and/or mammary epithelium.

Topics: Animals; Bone and Bones; Bone Neoplasms; Breast Neoplasms; Cell Transformation, Neoplastic; Humans; Osteoclasts; Osteoporosis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Signal Transduction

Bone-morphogenetic proteins (BMPs) play an important role in development and many cellular processes. However, their functional role in the development and progression of breast cancer is not clearly understood. In the present study, we performed a systematic expression analysis of the 14 types of BMPs in 10 human breast cancer cell lines. We found that bone morphogenetic protein 4 (BMP4) was one of the most frequently expressed BMPs. Furthermore, the expression level of BMP4 was maybe correlated with the metastatic potential of the cancer lines. Accordingly, overexpression of BMP4 in the breast cancer cell lines MCF-7 and MBA-MD-231 promoted the migration and invasion phenotypes of the cancer cells, whereas RNAi-mediated knockdown of BMP4 expression inhibited the migration and invasion activities of the cancer cells. To identify the important factors that may mediate the BMP4 functions in breast cancer cells, we analyzed a panel of cancer-related genes, and found that the expression of matrix metalloproteinase-1 (MMP-1) and C-X-C chemokine receptor type 4 (CXCR4) sharply increased at both the mRNA and protein levels in the breast cancer cells overexpressing BMP4. Interestingly, when breast cancer cells MDA-MB-231 or MCF-7 were co-cultured with the osteoblast-like cells MG63 to mimic a bone metastasis microenvironment, BMP4 did not exhibit any significant effect on the expression of OPG or RANKL, two important factors in bone remodeling. BMPs antagonists, Noggin, parallel inhibited breast cancer cell migration and invasion and induced bone remodeling. Taken together, our results strongly suggest that BMP4 may promote the migration and invasion of breast cancer cells, at least in part by up-regulating the expressions of MMP-1 and CXCR4. It is conceivable that novel therapeutics for breast cancer may be developed by targeting BMP4 signaling pathway and/or its important downstream mediators in breast cancer cells.

Topics: Activin Receptors; Bone Morphogenetic Protein 4; Breast Neoplasms; Carrier Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Coculture Techniques; Female; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Matrix Metalloproteinase 1; Neoplasm Invasiveness; Osteoblasts; Osteoprotegerin; Phenotype; RANK Ligand; Receptors, CXCR4; RNA Interference; RNA, Messenger; Smad Proteins; Time Factors; Transfection

Patients with locally advanced breast cancer treated with neoadjuvant chemotherapy are at risk of cancer treatment-induced bone loss and consequently of increased skeletal morbidity. In addition, this situation could be worsened by the fact that only a minority of patients with breast cancer have sufficient vitamin D. A comprehensive evaluation of bone homeostasis is critical in this context. We retrospectively evaluated the serum levels of calcium, vitamin D, TRAIL, RANK ligand (RANKL), Osteoprotegerin (OPG), Bone TRAP, CrossLaps and DKK1 in 77 patients (median age: 50 years; range 25-74), with locally advanced breast cancer treated in our institute with anthracyclines-taxane neoadjuvant chemotherapy (7 cycles of 21 days/each) between March 2007 and August 2008. Serum samples were collected before the first (baseline) and the last treatment cycle. Variations and correlations between biomarker levels were evaluated. At baseline, 79.5 % of patients had vitamin D insufficiency (<30 ng/ml), increasing to 97.4 % at the end of the neoadjuvant chemotherapy (p < 0.0001). Calcium and RANKL serum concentrations were also significantly decreased, while OPG was significantly increased, resulting in lower RANKL/OPG ratio. Calcium and vitamin D, RANKL and vitamin D and RANKL and OPG levels were significantly correlated (Spearman's coefficient r = 0.2721, p = 0.0006; r = 0.1916, p = 0.002; and r = -0.179, p = 0.03, respectively). Nearly all included patients suffered from vitamin D insufficiency by the end of the neoadjuvant chemotherapy with changes in the calcium/RANKL/OPG axis that are evocative of deregulation of a functional regulatory mechanism. Further studies are needed to determine how drugs modulate this regulatory mechanism to preserve bone homeostasis in patients with breast cancer.

Topics: Acid Phosphatase; Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Biomarkers; Bone and Bones; Breast Neoplasms; Calcium; Carcinoma, Ductal, Breast; Chemotherapy, Adjuvant; Collagen; Cyclophosphamide; Docetaxel; Epirubicin; Female; Fluorouracil; Humans; Intercellular Signaling Peptides and Proteins; Isoenzymes; Middle Aged; Neoadjuvant Therapy; Osteoprotegerin; Peptide Fragments; Prevalence; RANK Ligand; Retrospective Studies; Statistics, Nonparametric; Tartrate-Resistant Acid Phosphatase; Taxoids; TNF-Related Apoptosis-Inducing Ligand; Vitamin D; Vitamin D Deficiency

Tumor cells in bone can induce the activation of osteoclasts, which mediate bone resorption and release of growth factors and calcium from the bone matrix, resulting in a cycle of tumor growth and bone breakdown. Targeting the bone microenvironment by the inhibition of RANKL, an essential mediator of osteoclast function, not only prevents tumor-induced osteolysis but also decreases skeletal tumor burden in preclinical models. The inhibition of skeletal tumor progression after the inhibition of osteoclasts is via interruption of the "vicious cycle" of tumor/bone interactions. The majority of breast cancer patients at risk for bone metastases harbor estrogen receptor-positive (ER+) tumors. We developed a mouse model for ER+ breast cancer bone metastasis and evaluated the effect of RANKL inhibition on tumor-induced osteolysis and skeletal tumor growth both alone and in combination with tamoxifen. Luciferase-labeled MCF-7 cells (MCF-7Luc) formed metastatic foci in the hind limbs following intracardiac injection and caused mixed osteolytic/osteoblastic lesions. RANKL inhibition by OPG-Fc treatment blocked osteoclast activity and prevented tumor-induced osteolysis, as well as caused a marked decrease in skeletal tumor burden. Tamoxifen as a single agent reduced MCF-7Luc tumor growth in the hind limbs. In a combination experiment, OPG-Fc plus tamoxifen resulted in significantly greater tumor growth inhibition than either single agent alone. Histologic analysis revealed a decrease in the proliferation of tumor cells by both single agents, which was enhanced in the combination treatment. Upon treatment with OPG-Fc alone or in combination with tamoxifen, there was a complete absence of osteolytic lesions, demonstrating the ability of RANKL inhibition to prevent skeletal related morbidity in an ER+ model. The combination approach of targeting osteoclasts and the bone microenvironment by RANKL inhibition and the tumor directly via hormonal therapy may provide additional benefit to reducing skeletal tumor progression in ER+ breast cancer patients.

Topics: Animals; Apoptosis; Bone Density Conservation Agents; Bone Neoplasms; Breast Neoplasms; Disease Models, Animal; Female; Humans; MCF-7 Cells; Mice; Mice, Nude; Osteoclasts; Osteoprotegerin; RANK Ligand; Receptors, Estrogen; Selective Estrogen Receptor Modulators; Tamoxifen; Tumor Microenvironment

The aim of the present study was to evaluate correlations between serum osteocalcin, osteoprotegerin and NTX (Cross-linked N-telopeptides of Type I Collagen) and urinary NTX in breast and lung cancer patients with bone metastases. These four markers are considered to have important roles in bone formation, resorption and metastases.. Four markers were determined in the sera of 60 breast cancer and 21 lung cancer patients and healthy controls (n=30). Serum levels were studied using ELISA and EIA.. The median levels of serum osteoprotegerin (p<0.001) and osteocalcin (p=0.003) were higher in patients. Significant correlations were observed between the serum NTX-osteocalcin (r=0.431; p<0.001), serum NTX- osteoprotegerin (r=0.42; p=0.003) and serum NTX - urine NTX (r=0.255; p=0.022).. We conclude that osteocalcin, osteoprotegerin and NTX are independent diagnostic tools. Due to the ease of urine collection, urine NTX may be applied routinely to allow early detection of bone metastases and indicate progression of the disease.

Topics: Adult; Aged; Biomarkers, Tumor; Bone Neoplasms; Breast Neoplasms; Case-Control Studies; Collagen Type I; Female; Humans; Lung Neoplasms; Male; Middle Aged; Osteocalcin; Osteoprotegerin; Peptides

Osteoprotegerin (OPG) is a secreted member of the tumor necrosis factor (TNF) receptor superfamily that binds to the ligand for receptor activator of nuclear factor κB (RANKL) and inhibits bone resorption. OPG can also bind and inhibit the activity of the TNF-related apoptosis-inducing ligand (Apo2L/TRAIL), raising the possibility that the anticancer efficacy of soluble Apo2L/TRAIL may be abrogated in the bone microenvironment where OPG expression is high. In this study we used a murine model of breast cancer growth in bone to evaluate the efficacy of recombinant soluble Apo2L/TRAIL against intratibial tumors that were engineered to overexpress native full-length human OPG. In vitro, OPG-overexpressing breast cancer cells were protected from Apo2L/TRAIL-induced apoptosis, an effect that was reversed with the addition of soluble RANKL or neutralizing antibodies to OPG. In vivo, mice injected intratibially with cells containing the empty vector developed large osteolytic lesions. In contrast, OPG overexpression preserved the integrity of bone and prevented breast cancer-induced bone destruction. This effect was due primarily to the complete absence of osteoclasts in the tibias of mice inoculated with OPG-transfected cells, confirming the biologic activity of the transfected OPG in vivo. Despite the secretion of supraphysiologic levels of OPG, treatment with Apo2L/TRAIL resulted in strong growth inhibition of both empty vector and OPG-overexpressing intratibial tumors. While Apo2L/TRAIL-induced apoptosis may be abrogated in vitro by OPG overexpression, the in vivo anticancer efficacy of recombinant soluble Apo2L/TRAIL is retained in the bone microenvironment even in the presence of biologically active OPG at supraphysiologic concentrations.

Topics: Animals; Antineoplastic Agents; Apoptosis; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Cytoprotection; Female; Humans; Mice; Mice, Nude; Osteoprotegerin; Solubility; Tibia; TNF-Related Apoptosis-Inducing Ligand; Transfection; Treatment Outcome; Xenograft Model Antitumor Assays

Osteoprotegerin (OPG) is a decoy receptor of the receptor activator of nuclear factor-κB ligand (RANK-L) and plays an important role in the formation of metastatic bone lesions. We evaluated the usefulness of circulating OPG and RANK-L for the detection of bone metastases. We enrolled 143 individuals in the study: 30 healthy donors (HD) and 113 breast cancer patients. Among patients, 49 had no evidence of disease (NEDP), 54 had bone metastases (BMP) at first diagnosis, and 10 had visceral metastases (VMP). Both transcripts were determined in peripheral blood samples using quantitative PCR. Receiver operating characteristic (ROC) curve analysis was used to calculate the diagnostic accuracy of OPG, RANK-L, CEA and CA15-3. OPG and RANK-L median values were significantly lower in BMP (median 0.5, range 0.1-5.7, p<0.001 and median 0.5, range 0.1-4.5, p=0.024, respectively) compared to NEDP (median 1.7, range 0.4-8.9 and median 0.8, range 0.2-3.8, respectively), regardless of the number and type of bone lesions or the presence of visceral metastases. The area under the ROC curve (NEDP vs. BMP) was higher for OPG (82.5, 95% CI 74.5-90.6) than for RANK-L (69.2, 95% CI 59.0-79.40). Specificity for OPG was 87.7% (95% CI 75.7-94.2) and sensitivity was 74.1% (95% CI 60.4-85.0), both values increasing when considered together with CEA and CA15-3. For VMP, OPG and RANK-L were expressed in only one patient. Our results highlight the potentially important role of circulating OPG in the diagnosis of bone metastases. A confirmatory study on a larger case series is ongoing.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers; Bone Neoplasms; Breast Neoplasms; Breast Neoplasms, Male; Carcinoembryonic Antigen; Case-Control Studies; Cell Line, Tumor; Female; HT29 Cells; Humans; Male; Middle Aged; Osteoprotegerin; RANK Ligand; Retrospective Studies; RNA, Messenger; Sensitivity and Specificity

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

Bone metastasis is a complication occurring in up to 70% of advanced breast cancer patients. The estrogen receptor-related receptor alpha (ERRα) has been implicated in breast cancer and bone development, prompting us to examine whether ERRα may function in promoting the osteolytic growth of breast cancer cells in bone. In a mouse xenograft model of metastatic human breast cancer, overexpression of wild-type ERRα reduced metastasis, whereas overexpression of a dominant negative mutant promoted metastasis. Osteoclasts were directly affected and ERRα upregulated the osteoclastogenesis inhibitor, osteoprotegerin (OPG), providing a direct mechanistic basis for understanding how ERRα reduced breast cancer cell growth in bone. In contrast, ERRα overexpression increased breast cancer cell growth in the mammary gland. ERRα-overexpressing primary tumors were highly vascularized, consistent with an observed upregulation of angiogenic growth factor, the VEGF. In support of these findings, we documented that elevated expression of ERRα mRNA in breast carcinomas was associated with high expression of OPG and VEGF and with disease progression. In conclusion, our results show that ERRα plays a dual role in breast cancer progression in promoting the local growth of tumor cells, but decreasing metastatic growth of osteolytic lesions in bone.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Carcinoma; Cell Line, Tumor; ERRalpha Estrogen-Related Receptor; Female; Humans; Mice; Mice, Inbred BALB C; Neovascularization, Pathologic; Osteoprotegerin; Receptors, Estrogen; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

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

The development and spread of tumors is associated with the ability of malignant cells to avoid detection and subsequent elimination by the immune system, to grow in non-native sites and to avoid programmed or induced cell death. In addition to the well-described role of osteoprotegerin in the regulation of bone turnover, there is an emerging evidence that osteoprotegerin may have an additional function due to its ability to bind and inhibit the members of the tumor necrosis factor (TNF)-superfamily, such as TNF-alpha and TNF-related apoptosis inducing ligand (TRAIL). We have shown that the breast cancer cell line MDA-MB-231 produces a sufficient amount of osteoprotegerin to bind TRAIL, resulting in an upregulation of receptor activator factor kappa B ligand (RANKL) expression. In conclusion, the presence of osteoprotegerin, as secreted by this cell line, acting as a paracrine factor, could affect breast cancer RANKL production inducing an enhancement of osteolysis and the perpetuation of a vicious cycle. A better understanding of the complex tumor cell-host cell interactions in the bone microenvironment, and of the autocrine and paracrine effects of the secreted (from tumor cells) and released (from bone matrix) factors may facilitate development of effective strategies to inhibit disease progression.

Topics: Bone and Bones; Breast Neoplasms; Cell Line, Tumor; Estrogens; Female; Humans; Osteolysis; Osteoprotegerin; Paracrine Communication; RANK Ligand; TNF-Related Apoptosis-Inducing Ligand; Up-Regulation

Breast cancer has a propensity to metastasize to bone, thus causing pathological fractures. Bisphosphonates are established drugs in the treatment of bone metastasis that inhibit osteoclast activity and interrupt the vicious cycle of osteoclast-tumor cell interactions. We evaluated the direct effects of zoledronic acid on estrogen receptor (ER)-negative MDA-MB-231 and ER-positive MCF-7 breast cancer cells. While zoledronic acid (100 microM) inhibited MDA-MB-231 cell proliferation after 72 h, and induced apoptosis via activation of caspase-3 and -7, it had only minor effects on MCF-7 cells. In addition, zoledronic acid induced apoptosis by up-regulating TNF-related apoptosis-inducing ligand (TRAIL) in MDA-MB-231 cells (p<0.01), but had no effect on the expression of its decoy receptor osteoprotegerin (OPG). In MCF-7 cells, both cytokines were suppressed by zoledronic acid. In conclusion, zoledronic acid enhanced the TRAIL-to-OPG ratio in TRAIL-sensitive MDA-MB-231 cells, indicating that the TRAIL/OPG cytokine system is a bisphosphonate-responsive target in breast cancer.

Topics: Apoptosis; Bone Density Conservation Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Diphosphonates; Female; Humans; Imidazoles; Osteoprotegerin; RNA, Messenger; TNF-Related Apoptosis-Inducing Ligand; Zoledronic Acid

Skeletal disorders are a common complication of breast cancer and will be found in the vast majority of women with metastatic disease. Our study showed that rosmarinic acid (RA) could inhibit the migration of MDA-MB-231BO human bone-homing breast cancer cells dose-dependently. Furthermore, in ST-2 murine bone marrow stromal cells cultured with RA there was a significant and dose-dependent increase in alkaline phosphatase (ALP) activity, with the number and size of mineralized nodules increasing. According to Western blot and quantitative real-time PCR assay, RA may inhibit bone metastasis from breast carcinoma mainly via the pathway of the receptor activator of NF kappaB ligand (RANKL)/RANK/osteoprotegerin (OPG) and by simultaneously suppressing the expression of interleukin-8 (IL-8). RA may thus be a good candidate for a new therapeutic approach in bone metastasis from breast carcinoma.

Topics: Alkaline Phosphatase; Animals; Antineoplastic Agents, Phytogenic; Blotting, Western; Bone and Bones; Bone Marrow; Bone Neoplasms; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cinnamates; Depsides; Dose-Response Relationship, Drug; Female; Humans; Interleukin-8; Mice; NF-kappa B; Osteoprotegerin; Phytotherapy; Plant Extracts; Prunella; RANK Ligand; Reverse Transcriptase Polymerase Chain Reaction; Rosmarinic Acid; Stromal Cells

Vitamin D exerts antiproliferative, prodifferentiation, and proapoptotic effects on nonclassic target tissues such as breast. Blood levels of 25-hydroxyvitamin D [25(OH)D], the most sensitive indicator of vitamin D status, are inversely correlated with breast cancer risk; however, a causal relationship between vitamin D deficiency and breast cancer growth in bone has not been assessed. We examined the effect of vitamin D deficiency on the intraskeletal growth of the human breast cancer cell line MDA-MB-231-TxSA in a murine model of malignant bone lesions. Subsets of mice were treated concurrently with osteoprotegerin (OPG) to abrogate bone resorption. Outcomes were assessed by repeated radiographic and end-point micro-computed tomography and histologic analyses. Mice weaned onto a vitamin D-free diet developed vitamin D deficiency within 4 weeks [mean +/- SE serum 25(OH)D: 11.5 +/- 0.5 nmol/L], which was sustained throughout the study and was associated with secondary hyperparathyroidism and accelerated bone turnover. Osteolytic lesions appeared earlier and were significantly larger in vitamin D-deficient than in vitamin D-sufficient mice after 2 weeks (radiographic osteolysis: +121.5%; histologic tumor area: +314%; P < 0.05). Although OPG treatment reduced the size of radiographic osteolyses and tumor area in both groups, tumors remained larger in OPG-treated vitamin D-deficient compared with OPG-treated vitamin D-sufficient mice (0.53 +/- 0.05 mm(2) versus 0.19 +/- 0.05 mm2; P < 0.05). We conclude that vitamin D deficiency promotes the growth of human breast cancer cells in the bones of nude mice. These effects are partly mediated through secondary changes in the bone microenvironment, along with direct effects of vitamin D on tumor growth.

Topics: Animals; Bone Neoplasms; Bone Resorption; Breast Neoplasms; Calcifediol; Calcitriol; Cell Growth Processes; Cell Line, Tumor; Female; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Osteoprotegerin; Steroid Hydroxylases; Transplantation, Heterologous; Vitamin D; Vitamin D Deficiency; Vitamin D3 24-Hydroxylase

Hematopoietic growth factors are used to reverse chemotherapy-induced leukopenia. However, some factors such as granulocyte macrophage colony-stimulating factor (GM-CSF) induce osteoclast-mediated bone resorption that can promote cancer growth in the bone. Accordingly, we evaluated the ability of GM-CSF to promote bone metastases of breast cancer or prostate cancer in a mouse model of chemotherapy-induced leukopenia. In this model, GM-CSF reversed cyclophosphamide-induced leukopenia but also promoted breast cancer and prostate cancer growth in the bone but not in soft tissue sites. Bone growth was associated with the induction of osteoclastogenesis, yet in the absence of tumor GM-CSF, it did not affect osteoclastogenesis. Two osteoclast inhibitors, the bisphosphonate zoledronic acid and the RANKL inhibitor osteoprotegerin, each blocked GM-CSF-induced tumor growth in the bone but did not reverse the ability of GM-CSF to reverse chemotherapy-induced leukopenia. Our findings indicate that it is possible to dissociate the bone-resorptive effects of GM-CSF, to reduce metastatic risk, from the benefits of this growth factor in reversing leukopenia caused by treatment with chemotherapy.

Topics: Animals; Antineoplastic Agents, Alkylating; Bone Density Conservation Agents; Bone Neoplasms; Breast Neoplasms; Cyclophosphamide; Diphosphonates; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Imidazoles; Leukopenia; Male; Mice; Mice, Nude; Osteoclasts; Osteoprotegerin; Prostatic Neoplasms; RANK Ligand; Zoledronic Acid

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

Most patients with advanced breast cancer develop osteolytic bone metastases, which have numerous complications. Because current therapies are not curative, new treatments are needed. Conditionally replicating adenoviruses (CRAds) are anticancer agents designed to infect and lyse tumor cells. However, in spite of their promise as selective cancer therapeutics, replicating adenoviruses have shown limited efficacy in the clinical setting. We hypothesized that a CRAd armed with osteoprotegerin (OPG) would eradicate bone metastases of breast cancer both directly, by oncolysis, and indirectly, by inhibiting osteoclastic bone resorption, and thus reducing the tumor burden. We constructed an armed CRAd (Ad5-Δ24-sOPG-Fc-RGD) by replacing viral E3B genes with a fusion of the ligand-binding domains of OPG and the Fc portion of human IgG1. Conditional replication was conferred by a 24-base pair deletion within E1A (Δ24), which prevents the binding of E1A to the retinoblastoma tumor suppressor/cell cycle regulator protein and limits replication in normal cells. Enhanced infection of cells expressing low levels of the primary Ad5 receptor was conferred by incorporating an arginine-glycine-aspartic acid (RGD) peptide sequence into the fiber knob to mediate binding to α(v) integrins. After characterization of the armed CRAd, we demonstrated that infection of breast cancer cells by Ad5-Δ24-sOPG-Fc-RGD both killed the infected cells by oncolysis and inhibited the formation of osteoclasts in an in vitro co-culture model. In a murine model of osteolytic bone metastases of breast cancer, the CRAd armed with shortened OPG (sOPG)-Fc reduced tumor burden in the bone and inhibited osteoclast formation more effectively than an unarmed CRAd.

Topics: Adenoviridae; Animals; Bone Neoplasms; Breast Neoplasms; Carrier Proteins; Cell Line, Tumor; Female; Humans; Mice; Osteoprotegerin; Tumor Burden; Virus Replication

Breast cancer frequently metastasizes to bone, where tumor cells induce osteoclasts to locally destroy bone. During bone resorption, growth factors are locally released that may support bone metastatic growth. Differently from most other tissues, drugs that can limit local turnover, such as bisphosphonates and osteoprotegerin (OPG), are available for bone. We examined the hypothesis that inhibition of bone resorption by two different mechanisms may also affect the growth of cancer cells in bone. For this, we tested the effects of high doses of OPG and zoledronic acid (ZOL) on progression of MDA-231-B/Luc+ breast cancer cells in the bone microenvironment using whole body bioluminescent reporter imaging (BLI). Both treatments significantly inhibited the development of radiographically detectable osteolytic lesions. Histologic examination corroborated the radiographic findings, showing that both treatments preserved the integrity of bone trabeculae and prevented bone destruction (significantly higher trabecular bone volumes vs. vehicle). However, whereas practically no TRAcP-positive osteoclasts were observed in tibiae preparations of animals treated with Fc-OPG, TRAcP-positive osteoclasts were still present in the animals treated with ZOL. Intra-bone tumor burden was reduced with ZOL and Fc-OPG treatment. Although there appeared to be a trend for less overall total tumor burden upon treatment with both compounds, this was not significant as assessed by BLI and histomorphometric analysis due to the extramedullary growth of cancer cells which was not affected by these treatments. Collectively, anti-resorptive agents with different mechanisms of action - ZOL and OPG - significantly reduced cancer-induced osteolysis and intra-osseous tumor burden, but failed to restrain local tumor growth. However, interference with the bone micro-environmental growth support could still be of therapeutic relevance when given to patients early in the course of bone metastatic disease.

Topics: Acid Phosphatase; Animals; Bone and Bones; Bone Resorption; Breast Neoplasms; Cell Count; Cell Line, Tumor; Cell Proliferation; Diphosphonates; Female; Humans; Imidazoles; Isoenzymes; Luminescent Measurements; Mice; Mice, Inbred BALB C; Mice, Nude; Osteoclasts; Osteolysis; Osteoprotegerin; Radiography; Tartrate-Resistant Acid Phosphatase; Whole Body Imaging; Zoledronic Acid

Osteoprotegerin (OPG) is a decoy receptor for receptor activator of NF-kappaB ligand (RANKL) and TNF-related apoptosis-inducing ligand (TRAIL). While RANKL is essential for osteoclastogenesis and facilitates breast cancer migration into bone, TRAIL promotes breast cancer apoptosis. We analyzed the expression of OPG and TRAIL and its modulation in estrogen receptor-positive MCF-7 cells and receptor-negative MDA-MB-231 cells. In both cells, OPG mRNA levels and protein secretion were dose- and time-dependently enhanced by interleukin (IL)-1beta and suppressed by dexamethasone. In contrast to MCF-7 cells, MDA-MB-231 abundantly expressed TRAIL mRNA, which was enhanced by IL-1beta and inhibited by dexamethasone. TRAIL activated pro-apoptotic caspase-3, -7, and poly-ADP-ribose polymerase and decreased cell numbers of MDA-MB-231, but had no effect on MCF-7 cells. Gene silencing siRNA directed against OPG resulted in a 31% higher apoptotic rate compared to non-target siRNA-treated MDA-MB-231 cells. Furthermore, TRAIL induced significantly less apoptosis in cells cultured in conditioned media (containing OPG) compared to cells exposed to TRAIL in fresh medium lacking OPG (P < 0.01) and these protective effects were reversed by blocking OPG with its specific ligand RANKL (P < 0.05). The association between cancer cell survival and OPG production by MDA-MB-231 cells was further supported by the finding, that modulation of OPG secretion using IL-1beta or dexamethasone prior to TRAIL exposure resulted in decreased and increased rate of apoptosis, respectively (P < 0.05). Thus, OPG secretion by breast cancer cells is modulated by cytokines and dexamethasone, and may represent a critical resistance mechanism that protects against TRAIL-induced apoptosis.

Topics: Antineoplastic Agents, Hormonal; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Dexamethasone; Drug Resistance, Neoplasm; Female; Humans; Interleukin-1beta; Osteoprotegerin; TNF-Related Apoptosis-Inducing Ligand

Angiogenesis, the sprouting of capillaries from existing blood vessels, is central to tumour growth and progression, however the molecular regulation of this process remains to be fully elucidated. The secreted glycoprotein osteoprotegerin (OPG) is one potential pro-angiogenic factor, and clinical studies have demonstrated endothelial cells within a number of tumour types to express high levels of OPG compared to those in normal tissue. Additionally, OPG can increase endothelial cell survival, proliferation and migration, as well as induce endothelial cell tube formation in vitro. This study aims to elucidate the processes involved in the pro-angiogenic effects of OPG in vitro, and also how OPG levels may be regulated within the tumour microenvironment.. It has previously been demonstrated that OPG can induce tube formation on growth factor reduced matrigel. In this study, we demonstrate that OPG enhances the pro-angiogenic effects of VEGF and that OPG does not stimulate endothelial cell tube formation through activation of the VEGFR2 receptor. We also show that cell contact between HuDMECs and the T47D breast cancer cell line increases endothelial cell OPG mRNA and protein secretion levels in in vitro co-cultures. These increases in endothelial cell OPG secretion were dependent on alphanubeta3 ligation and NFkappaB activation. In contrast, the pro-angiogenic factors VEGF, bFGF and TGFbeta had no effect on HuDMEC OPG levels.. These findings suggest that the VEGF signalling pathway is not involved in mediating the pro-angiogenic effects of OPG on endothelial cells in vitro. Additionally, we show that breast cancer cells cause increased levels of OPG expression by endothelial cells, and that direct contact between endothelial cells and tumour cells is required in order to increase endothelial OPG expression and secretion. Stimulation of OPG secretion was shown to involve alphanubeta3 ligation and NFkappaB activation.

Topics: Analysis of Variance; Breast Neoplasms; Cell Communication; Cell Line, Tumor; Endothelial Cells; Female; Humans; Integrin alphaVbeta3; Neovascularization, Pathologic; NF-kappa B; Osteoprotegerin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

Bone metastasis is mediated by complex interactions between tumor cells and resident stromal cells in the bone microenvironment. The functions of metalloproteinases in organ-specific metastasis remain poorly defined despite their well-appreciated role in matrix degradation and tumor invasion. Here, we show a mechanism whereby two distinct metalloproteinases, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS1) and matrix metalloproteinase-1 (MMP1), orchestrate a paracrine signaling cascade to modulate the bone microenvironment in favor of osteoclastogenesis and bone metastasis. Proteolytic release of membrane-bound epidermal growth factor (EGF)-like growth factors, including Amphiregulin (AREG), heparin-binding EGF (HB-EGF), and transforming growth factor alpha (TGFalpha) from tumor cells suppress the expression of osteoprotegerin (OPG) in osteoblasts and subsequently potentiate osteoclast differentiation. EGF receptor (EGFR) inhibitors block osteolytic bone metastasis by targeting EGFR signaling in bone stromal cells. Furthermore, elevated MMP1 and ADAMTS1 expression is associated with increased risk of bone metastasis in breast cancer patients. This study established MMP1 and ADAMTS1 in tumor cells, as well as EGFR signaling in osteoblasts, as promising therapeutic targets for inhibiting bone metastasis of breast cancer.

Topics: ADAM Proteins; ADAMTS1 Protein; Animals; Bone and Bones; Bone Neoplasms; Breast Neoplasms; Cell Differentiation; Cell Line; Cell Proliferation; Epidermal Growth Factor; Female; Gefitinib; Gene Expression; Gene Expression Regulation, Neoplastic; Gene Silencing; Matrix Metalloproteinase 1; Mice; Mice, Nude; Osteoblasts; Osteoprotegerin; Protein Kinase Inhibitors; Quinazolines; RANK Ligand; Signal Transduction

Most breast cancer metastases in bone form osteolytic lesions, but the mechanisms of tumor-induced bone resorption and destruction are not fully understood. Although it is well recognized that Wnt/beta-catenin signaling is important for breast cancer tumorigenesis, the role of this pathway in breast cancer bone metastasis is unclear. Dickkopf1 (Dkk1) is a secreted Wnt/beta-catenin antagonist. In the present study, we demonstrated that activation of Wnt/beta-catenin signaling enhanced Dkk1 expression in breast cancer cells and that Dkk1 overexpression is a frequent event in breast cancer. We also found that human breast cancer cell lines that preferentially form osteolytic bone metastases exhibited increased levels of Wnt/beta-catenin signaling and Dkk1 expression. Moreover, we showed that breast cancer cell-produced Dkk1 blocked Wnt3A-induced osteoblastic differentiation and osteoprotegerin (OPG) expression of osteoblast precursor C2C12 cells and that these effects could be neutralized by a specific anti-Dkk1 antibody. In addition, we found that breast cancer cell conditioned media were able to block Wnt3A-induced NF-kappaB ligand reduction in C2C12 cells. Finally, we demonstrated that conditioned media from breast cancer cells in which Dkk1 expression had been silenced via RNAi were unable to block Wnt3A-induced C2C12 osteoblastic differentiation and OPG expression. Taken together, these results suggest that breast cancer-produced Dkk1 may be an important mechanistic link between primary breast tumors and secondary osteolytic bone metastases.

Topics: beta Catenin; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Humans; Intercellular Signaling Peptides and Proteins; NF-kappa B; Osteoblasts; Osteolysis; Osteoprotegerin; RANK Ligand; Up-Regulation; Wnt Proteins; Wnt3 Protein; Wnt3A Protein

The majority of human breast cancers and in addition most breast-cancer cell lines express gonadotropin-releasing hormone (GnRH) receptors. Their proliferation and in addition their bone-directed invasion is time- and dose-dependently reduced by GnRH. Osteolytic metastases are characteristic for breast cancer-derived metastasis. Since the osteolytic activity depends on the receptor activator of nuclear factor-kappaB (NFkappaB) ligand (RANKL)/osteoprotegerin (OPG) ratio, we analyzed RANKL and OPG expression in different breast-cancer cell lines.. Different human breast-cancer cell lines were tested for expression of GnRH receptor, OPG and RANKL. Using a co-culture system of breast-cancer cell lines and human primary osteoblasts (hOB), we analyzed the expression of OPG and RANKL in the GnRH receptor-positive breast-cancer cell line HCC70 co-cultured with or without hOB. In addition, we assessed the effects of GnRH analog treatment on OPG and RANKL mRNA and protein levels.. All tested breast-cancer cell lines were GnRH receptor-positive. The majority of these cell lines expressed OPG but not RANKL. The HCC70 breast-cancer cell line derived from an invasive ductal carcinoma with metastases was positive for both OPG and RANKL. The expression of RANKL by HCC70 cells was increased when co-cultured with hOB. Treatment with GnRH analogs reduced the expression of RANKL by HCC70 cells co-cultured with hOB. No effects were observed on breast cancer OPG expression.. These data show that the majority of human breast-cancer cell lines express OPG but not RANKL. The HCC70 breast-cancer cell line is RANKL-positive. Co-culture of HCC70 breast cancer cells with hOB increases RANKL expression. Activation of tumor GnRH receptors reduces RANKL expression. These experiments demonstrate that HCC70 breast cancer cells are able to activate osteoclasts directly via RANKL. The interaction between HCC70 breast cancer cells and osteoblasts induces osteoclastogenesis through an increase of RANKL expression. GnRH seems to play an important role by modulating the RANKL expression in HCC70 breast cancer cells.

Topics: Antineoplastic Agents, Hormonal; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Coculture Techniques; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Osteoblasts; Osteoprotegerin; RANK Ligand; Receptors, LHRH; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Triptorelin Pamoate

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

Breast cancer cells preferentially metastasize to bone, leading to the formation of primarily osteolytic lesions. Osteoprotegerin (OPG) plays multifactorial roles in the development of osteolytic bone metastases. An increase in the ratio of receptor activator of nuclear factor kappaB ligand (RANKL) to OPG increases osteoclastogenesis within the bone microenvironment. OPG also acts as a survival factor for cancer cells by protecting them from tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediated apoptosis. This study compares OPG production in vitro in a number of breast cancer cell lines exhibiting both differences in metastatic capacity and in preferential metastasis to bone. Our studies demonstrated that OPG expression by MDA-231, MDA-MET, and MDA-231/K cancer cells was directly correlated with bone specific homing and colonization potential but not with metastasis of cancer cells to other organs; both in IL-1 beta stimulated and control cells. We also demonstrated expression of other bone-related markers including type I collagen, osteocalcin, osteopontin, and Runx2 in these cells. However, the generally lower expression of these markers in the bone selective cell line MDA-MET suggested that increased OPG expression in the bone specific variant was not merely a consequence of enhanced osteomimicry by these cells but that it has a significant role in the metastatic process. Co-culture of breast cancer cells with osteoblastic cells (hFOB 1.19) led to an overall downregulation in OPG production, which was not affected by the bone homing and colonization potential of the cell lines, suggesting that OPG alone is not indicative of osteolytic bone activity by breast cancer cells.

Topics: Bone and Bones; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Coculture Techniques; Gene Expression Regulation, Neoplastic; Humans; Interleukin-1beta; Osteoblasts; Osteoprotegerin; RNA, Messenger

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-kappaB 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.

Topics: Animals; Apoptosis; Bone and Bones; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Disease Models, Animal; Female; Humans; Immunoglobulin Fc Fragments; Mice; Mice, Inbred BALB C; Mice, Nude; Osteoprotegerin; RANK Ligand; Recombinant Fusion Proteins; Transplantation, Heterologous

Estrogen regulates various cytokines and growth factors in estrogen receptor (ER)-positive human breast cancer. Receptor activator of NF-kappaB ligand (RANKL) is an essential cytokine for osteoclasts, whereas osteoprotegerin (OPG) is a soluble inhibitor for RANKL. We analyzed the regulation of the RANKL/OPG system by estrogens and androgens in the ER-positive breast cancer cell line MCF-7 and the ER-negative breast cancer cell line MDA-MB-231. In MCF-7 cells, which predominantly express ER-alpha, 17beta-estradiol and testosterone dose-dependently decreased OPG mRNA levels and protein secretion by 70 and 65%, respectively (p<0.0001 by ANOVA). The inhibition of OPG production by 17beta-estradiol and testosterone was specifically prevented by the pure anti-estrogen ICI 182,780, and the testosterone effect was prevented by an aromatase inhibitor. In conclusion, 17beta-estradiol suppressed OPG production by human breast cancer cell lines in a dose-dependent and specific manner, indicating that the RANKL/OPG cytokine system is an estrogen-responsive target in breast cancer.

Topics: Breast Neoplasms; Cell Line, Tumor; Dose-Response Relationship, Drug; Down-Regulation; Estradiol; Estrogen Receptor alpha; Gene Expression Regulation, Neoplastic; Humans; Osteoprotegerin

Human breast cancer frequently metastasizes to bone, and effective therapies for patients with bone metastasis are required. However, the molecular mechanism for the bone metastasis of human breast cancer has not yet been fully elucidated. The present study aimed to evaluate the importance of active osteoclasts and bone-derived insulin-like growth factors (IGFs) for the survival and growth of breast cancer cells in bone. Human breast cancer cell line MCF-7 cells were injected into human adult bone (HAB) implanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. The mice were then treated with recombinant human osteoclastogenesis inhibitory factor/osteoprotegerin (rhOCIF/OPG), a decoy receptor for receptor activator of NF-kappaB ligand (RANKL), or an anti-human IGF monoclonal antibody. Histomorphometric analyses revealed that both treatments significantly decreased the tumor area of MCF-7 cells in cross-sections of the implanted HAB to about 30% of the tumor area in control mice, but had no effect on the growth of subcutaneously injected MCF-7 cells. Consistent with the results for the tumor area in HAB, there were fewer osteoclasts in the implanted HAB in rhOCIF/OPG-treated mice than in vehicle-treated mice. However, treatment with the anti-human IGF monoclonal antibody had no effect on the number of osteoclasts in HAB. The results indicate that the active osteoclasts induced by RANKL and the IGFs released as a result of bone resorption by these osteoclasts play crucial roles in the survival and growth of human breast cancer cells in bone and suggest that neutralization of bone-derived IGFs will be effective in preventing the development of bone tumors in breast cancer patients.

Topics: Animals; Bone Neoplasms; Bone Transplantation; Breast Neoplasms; Cell Line, Tumor; Female; Humans; Mice; Mice, Inbred NOD; Mice, SCID; Osteoclasts; Osteoprotegerin; Somatomedins; Transplantation, Heterologous

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

Inhibition of bone resorption either by bisphosphonate (BP) treatment or by blocking RANKL signalling with osteoprotegerin (OPG) has been shown to reduce tumour burden in bone and inhibit bone destruction in murine xenograft models of breast cancer. However, whether the anti-tumour effect of OPG or BP in bone is mediated by inhibition of bone resorption or by direct effects on tumour cells is uncertain. The current study is designed to investigate anti-tumour effects of OPG and ibandronate (IBN), dosed alone or in combination, on tumour growth to determine if there is experimental support for combination treatments and to provide evidence for the presence of direct anti-tumour effects. To this aim, 10 microl (5 x 10(6) cells/ml) of the bone-seeking MDA-MB-231 (Tx-SA) cell line was injected intra-tibially into nude mice. After 10 days, when the tumours were evident radiologically, mice were treated with vehicle, OPG (1 mg/kg/day), ibandronate (IBN) (160 microg/kg/day) or IBN and OPG at the same doses (IBN+OPG) for a week, and the effects of each treatment on lytic lesions, tumour cell growth, cell apoptosis and proliferation were measured by radiography, immunohistochemistry and histomorphometry. Compared to vehicle controls, in vivo treatment with OPG, IBN, or IBN+OPG, each prevented the expansion of osteolytic bone lesions (increase in lytic lesion area day 10 to day 17: OPG -3.2%, IBN 6.6%, IBN+OPG 3.6%, Vehicle 232.5%; p<0.01). Treatment with OPG, IBN or IBN+OPG each produced similar reductions in tumour area relative to vehicle-treated mice (OPG 52%, IBN 54%, IBNp and OPG 48%, p<0.01 vs. vehicle) OPG and IBN alone and in combination each produced a similar increase in cancer cell apoptosis (OPG 330%, IBN 342%, IBN and OPG 347%, p<0.01 vs. vehicle) and a decrease in cancer cell proliferation (OPG 59%, IBN 62%, IBN and OPG 58%, p<0.05 vs. vehicle). Our findings indicate that (i) combined treatment with OPG and a bisphosphonate is not significantly more effective than either agent alone; and that (ii) inhibition of bone resorption, rather than direct anti-tumour action, mediates the effects of these agents on tumour growth in this in vivo model.

Topics: Animals; Apoptosis; Bone Density Conservation Agents; Bone Neoplasms; Bone Resorption; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Diphosphonates; Drug Therapy, Combination; Female; Ibandronic Acid; Mice; Mice, Inbred BALB C; Mice, Nude; Osteoprotegerin; Tibia; Xenograft Model Antitumor Assays

The role of receptor activator of nuclear factor-kappaB ligand (RANKL)/osteoprotegerin (OPG) system, and osteopontin (OPN) was studied in patients with solid tumors metastatic to the bone in relation to the type of malignancy and the neoplastic burden to the skeleton. Levels of soluble RANKL (sRANKL), OPG and OPN were assessed in 61 patients with breast, lung and prostate cancer with newly-diagnosed metastasis to the bone, in parallel with bone resorption [C-telopeptide of type-I collagen (CTX), tartrate-resistant acid phosphatase-5b (TRACP-5b)] and bone formation markers [bone-alkaline phosphatase (bALP), osteocalcin (OC), and C-terminal propeptide of collagen type-I (CICP)]. Patients had elevated serum levels of sRANKL, OPG, OPN, TRACP-5b, and bALP, and reduced OC levels compared to controls. OPG correlated with the extent of metastatic bone burden. Patients with breast and lung cancer shared increased levels of sRANKL, OPG, and OPN whereas prostate cancer patients had elevated values of OPG and bALP only. These results suggest that patients with solid tumors metastatic to the bone have severe disruption of the sRANKL/OPG axis. Breast and lung cancer seem to exert their osteolytic action through upregulation of the sRANKL/OPG system and OPN, whereas prostate cancer seems to provoke profound elevation of OPG levels only, thus leading to increased osteoblastic activity.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Bone and Bones; Bone Neoplasms; Bone Remodeling; Breast Neoplasms; Down-Regulation; Female; Humans; Lung Neoplasms; Male; Osteoblasts; Osteopontin; Osteoprotegerin; Prostatic Neoplasms; RANK Ligand; Skeleton; Up-Regulation

The skeleton is a major site of breast cancer metastases. High bone turnover increases risk of disease progression and death. However, there is no direct evidence that high bone turnover is causally associated with the establishment and progression of metastases. In this study, we investigate the effects of high bone turnover in a model of breast cancer growth in bone. Female nude mice commenced a diet containing normal (0.6%; 'Normal-Ca') or low (0.1%; 'Low-Ca') calcium content. Mice were concurrently treated with vehicle or osteoprotegerin (1 mg/kg/d s.c; n = 16 per group). Three days later (day 0), 50,000 Tx-SA cells (variant of MDA-MB-231 cells) were implanted by intratibial injection. On day 0, mice receiving Low-Ca had increased serum parathyroid hormone (PTH) and tartrate-resistant acid phosphatase 5b levels, indicating secondary hyperparathyroidism and high bone turnover, which was maintained until day 17. Osteoprotegerin increased serum PTH but profoundly reduced bone resorption. On day 17, in mice receiving Low-Ca alone, lytic lesion area, tumor area, and cancer cell proliferation increased by 43%, 24%, and 24%, respectively, compared with mice receiving Normal Ca (P < 0.01). Osteoprotegerin treatment completely inhibited lytic lesions, reduced tumor area, decreased cancer cell proliferation, and increased cancer cell apoptosis. Increased bone turnover, due to dietary calcium deficiency, promotes tumor growth in bone, independent of the action of PTH. Breast cancer patients frequently have low dietary calcium intake and high bone turnover. Treatment to correct calcium insufficiency and/or treatment with antiresorptive agents, such as osteoprotegerin, may be of benefit in the adjuvant as well as palliative setting.

Topics: Bone Neoplasms; Bone Resorption; Breast Neoplasms; Calcium; Calcium, Dietary; Cell Growth Processes; Cell Line, Tumor; Humans; Hyperparathyroidism; Osteoprotegerin; Parathyroid Hormone

The progression of cancer depends on the establishment of a tumour blood supply, and therefore tumour angiogenesis has been identified as a major target for new anticancer agents. Recent reports have suggested that osteoprotegerin (OPG) is involved in the control of endothelial cell survival through the inhibition of the activity of tumour necrosis factor- (TNF) related apoptosis-inducing ligand (TRAIL). The role of OPG in human tumour development and angiogenesis is currently unknown. In the present study we demonstrate the ability of OPG to support endothelial cell survival, as well as the formation of cord-like structures in vitro using a matrigel tubule formation assay. Investigation of various human cancers demonstrated endothelial OPG expression in 59% of malignant tumours (n=512), but in contrast, OPG was absent in endothelial cells associated with benign tumours and normal tissues (n=178). In a series of 400 breast tumours, endothelial OPG expression was associated with high tumour grade and certain histological types. Our data show a clear separation in endothelial OPG expression between malignant tumours and nonmalignant tissues, supporting a potential biological role for this molecule in the development and/or maintenance of the tumour vasculature. This is the first study to report the proangiogenic effects of OPG in vitro, as well as correlating expression of OPG by tumour endothelial cells with clinicopathological data in human tumours.

Topics: Breast Neoplasms; Cell Survival; Endothelial Cells; Female; Gene Expression Profiling; Glycoproteins; Humans; Immunohistochemistry; Neovascularization, Pathologic; Osteoprotegerin; Phenotype; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Osteoprotegerin (OPG) is involved in the regulation of bone turnover through binding to the receptor activator of nuclear factor kappaB ligand (RANKL), and has also been reported to be a potential survival factor for several different cell types. The survival effects are mediated through inhibition of the activity of tumour necrosis factor related apoptosis inducing ligand (TRAIL). Both breast and prostate cancer cells produce sufficient amounts of OPG to be protected against the effects of TRAIL in vitro.. To investigate the spatial expression of OPG, RANKL, and TRAIL in non-neoplastic breast tissue and breast cancer, and its relation with oestrogen receptor (ER) expression.. Forty breast cancers (20 ER+, 20 ER-) and five non-neoplastic breast tissue samples were stained with antibodies against OPG, RANKL, and TRAIL.. OPG was not expressed in non-neoplastic breast tissue except when colocalised with altered columnar epithelium. RANKL was expressed at the apical surface of luminal epithelial cells and TRAIL was expressed in myoepithelial cells. All three proteins were expressed in some breast cancers but showed no significant association with tumour type. OPG expression showed a significant positive correlation with ER expression (p = 0.011).. This is the first published study of the spatial expression of OPG, RANKL, and TRAIL in breast tissue and breast cancer. The localisation of each protein was specific and they were not colocalised. This specificity may provide a useful marker of functional differentiation in breast cancer; for example, TRAIL expression as a marker of myoepithelial differentiation.

Topics: Apoptosis Regulatory Proteins; Biomarkers, Tumor; Breast; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Carrier Proteins; Female; Glycoproteins; Humans; Membrane Glycoproteins; Neoplasm Invasiveness; Neoplasm Proteins; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Estrogen; Receptors, Progesterone; Receptors, Tumor Necrosis Factor; Retrospective Studies; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Receptor activator of nuclear factor kappabeta ligand (RANKL) has an important role in bone remodelling, and tumour necrosis factor related, apoptosis inducing ligand (TRAIL) can induce apoptosis in cancer cells. Their functions are linked by their interactions with osteoprotegerin (OPG).. To investigate the expression of RANKL and TRAIL in a large series of unselected breast cancers and to analyse the relations between these expressions and the expression of OPG, oestrogen receptor, and clinicopathological variables.. 395 breast cancers were sampled into tissue microarrays and immunohistochemistry undertaken for RANKL and TRAIL.. There was strong expression of RANKL in 14% of the cancers and strong expression of TRAIL in 30%. Expression of RANKL had a negative association with expression of oestrogen receptor (p = 0.036). Expression of TRAIL had a negative association with the Nottingham Prognostic Index (p = 0.021). There was a significant negative relation between expression of RANKL and TRAIL (p<0.005). Unsupervised cluster analysis produced a dendrogram that showed a clear division into two groups, and the expression of oestrogen receptor was significantly higher in one of those groups (p = 0.012).. There is apparent loss of expression of RANKL in 86% of breast cancers; those tumours that retain expression tend to be oestrogen receptor negative and of a high histological grade. There is strong expression of TRAIL in 30% of breast cancers and these tend to be of better prognostic type. These results may be important in the processes of metastasis to bone and the apoptotic cell death pathway in cancer.

Topics: Apoptosis Regulatory Proteins; Biomarkers, Tumor; Breast Neoplasms; Carrier Proteins; Cluster Analysis; Female; Glycoproteins; Humans; Membrane Glycoproteins; Neoplasm Invasiveness; Neoplasm Proteins; Osteoprotegerin; Prognosis; Protein Array Analysis; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Estrogen; Receptors, Tumor Necrosis Factor; Survival Analysis; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

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

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Bone Neoplasms; Breast Neoplasms; Cell Growth Processes; Cell Line, Tumor; Glycoproteins; Humans; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mice, Nude; Osteoclasts; Osteoprotegerin; Parathyroid Hormone-Related Protein; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; TNF-Related Apoptosis-Inducing Ligand; Transfection; Transplantation, Heterologous; Tumor Necrosis Factor-alpha

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

In addition to its role in bone turnover, osteoprotegerin (OPG) has been reported to bind to and inhibit Tumour necrosis factor-related apoptosis inducing ligand (TRAIL). TRAIL is produced in tumours by invading monocytes, inducing apoptosis in neoplastic cells sensitive to this cytokine. OPG production by tumour cells would therefore be a novel mechanism whereby cancer cells evade host defences and gain a growth advantage. In this study we show that OPG produced by breast cancer cells enhances tumour cell survival by inhibiting TRAIL-induced apoptosis. OPG expression by breast cancer cells (MDA-MB 436/231) grown in vitro was examined using PCR and ELISA, and the sensitivity of these cells to TRAIL was determined. The effects of OPG on TRAIL induced apoptosis was investigated by exposing MDA-MB 436 cells to TRAIL, in the presence or absence of OPG, followed by assessment of nuclear morphology. We found that the levels of OPG produced were sufficient to inhibit TRAIL-induced apoptosis, suggesting that OPG may play a role in tumour cell survival. We also examined the expression pattern of OPG in a selection of breast tumours (n=400) by immunohistochemistry, and related OPG expression to the clinico-pathological data for each tumour. OPG expression was found to be negatively correlated with increasing tumour grade. To our knowledge these results are the first to demonstrate that OPG can act as an endocrine survival factor for breast cancer cells, as well as reporting the expression patterns of OPG in a large cohort of human breast tumours.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Female; Glycoproteins; Humans; Immunohistochemistry; Membrane Glycoproteins; Osteoprotegerin; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

Advanced breast cancer is often associated with metastatic bone disease, causing a number of serious complications for the patients such as hypercalceamia, pain, nerve compression and fractures. The formation of bone metastases depends on complex interactions between tumour cells and the cells of the bone microenvironment, but the precise molecular mechanisms involved in the development of tumour-induced bone disease have not been identified. We have investigated the ability of bone marrow stromal cells (BMSC) isolated from breast cancer patients to generate osteoprotegerin (OPG), a molecule involved both in bone turnover and cell survival. The potential survival effects of OPG are mediated through binding to a member of the TNF super family, TNF-related Apoptosis Inducing Ligand (TRAIL), preventing association between TRAIL and its death-inducing receptors present on a number of tumour cell types. In the present report we show that bone marrow stromal cells isolated from breast cancer patients produce OPG when grown in culture. The levels of OPG present in BMSC conditioned medium is sufficient to protect breast cancer cells from undergoing TRAIL induced apoptosis. Our data suggest that bone-derived OPG may increase survival of breast cancer cells that reach the bone microenvironment as part of the metastatic process.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Bone Marrow Cells; Bone Neoplasms; Breast Neoplasms; Cell Survival; fas Receptor; Female; Glycoproteins; Humans; Ligands; Membrane Glycoproteins; Osteoprotegerin; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Stromal Cells; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Osteoprotegerin (OPG) is a potent antiresorptive molecule that binds NF-kappaB ligand, the final effector for osteoclastogenesis. OPG production is regulated by a number of cytokines and hormones. Osteopontin (OPN) is a secreted adhesive glycoprotein involved in tumour angiogenesis, and also a non-collagenous protein involved in bone turnover. OPN serum value is associated with tumour burden and survival in advanced breast cancer patients. The short-term effects of anastrozole on OPG and OPN serum values, and the usefulness of these analytes during follow-up were studied in 34 consecutive advanced breast cancer patients receiving anastrozole 1 mg/day. Blood samples were taken before treatment and at 2, 4, 8 and 12 weeks. OPG and OPN values were measured by ELISA. The results were analysed for all patients, and also separately for patients with (group A, 22 patients) and without (group B, 12 patients) bone metastasis. Whether the survival of all patients was related to their OPN serum values was also tested by placing patients into three groups (terciles) according to their baseline OPN values. No significant changes in OPG and OPN values were observed in the complete patient group. There was no difference in baseline OPG and OPN serum values between patients in groups A and B. In group A, a significant percentage increase in both OPG and OPN values from baseline was detected during treatment. No significant changes were reported for group B patients. Furthermore, in group A, a significant increase in both analytes was evident only for patients with progressive disease (PD). The Kaplan-Meier adjusted survival estimates for patients grouped according to tercile OPN values differed significantly (P = 0.001, log rank test). In conclusion, in the short term, anastrozole does not seem to affect OPG and OPN serum values in patients without bone disease. OPG and OPN appear to be useful predictors of the outcome of skeletal disease and elevated OPN values may be associated with short survival in advanced breast cancer patients.

Topics: Adult; Aged; Aged, 80 and over; Anastrozole; Antineoplastic Agents, Hormonal; Biomarkers, Tumor; Bone Neoplasms; Breast Neoplasms; Female; Glycoproteins; Humans; Middle Aged; Nitriles; Osteopontin; Osteoprotegerin; Postmenopause; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Sialoglycoproteins; Survival Analysis; Triazoles

Interleukin 8 (IL-8) is a member of the alpha chemokine family of cytokines originally identified as a neutrophil chemoattractant. Recently, we reported that elevated levels of IL-8, but not parathyroid hormone-related protein (PTHrP), correlated with increased bone metastasis in a population of human breast cancer cells. We hypothesized that IL-8 expression by breast cancer cells would either indirectly influence osteoclastogenesis via nearby stromal cells or directly influence osteoclast differentiation and activity. In the present study, we investigated the role of IL-8 in the process of osteoclast formation and bone resorption, which is associated with metastatic breast cancer. The addition of recombinant human (rh) IL-8 (10 ng/ml) to cultures of stromal osteoblastic cells stimulated both RANKL mRNA expression and protein production, with no effect on the expression of osteoprotegerin. In addition, rhIL-8 also directly stimulated the differentiation of human peripheral blood mononuclear cells into bone-resorbing osteoclasts. In these cultures, IL-8 was able to stimulate human osteoclast formation even in the presence of excess (200 ng/ml) RANK-Fc. The effect of IL-8 on osteoclasts and their progenitors was associated with the cell surface expression of the IL-8-specific receptor (CXCR1) on the cells. These results demonstrate a direct effect of IL-8 on osteoclast differentiation and activity. Together, these data implicate IL-8 in the osteolysis associated with metastatic breast cancer.

Topics: Animals; Bone Neoplasms; Bone Resorption; Breast Neoplasms; Cell Line, Tumor; Glycoproteins; Humans; Interleukin-8; Mice; Osteoclasts; Osteolysis; Osteoprotegerin; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Recombinant Proteins

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

Certain malignancies, including breast cancer, frequently metastasize to bone, where the tumor cells induce osteoclasts to locally destroy bone. Osteoprotegerin (OPG), a member of the tumor necrosis factor receptor family, is a negative regulator of osteoclast differentiation, activation, and survival. We tested the ability of recombinant OPG to inhibit tumor-induced osteoclastogenesis, osteolysis, and skeletal tumor burden in two animal models. In a syngeneic model, mouse colon adenocarcinoma (Colon-26) cells were injected into the left ventricle of mice. Treatment with OPG dose-dependently decreased the number and area of radiographically evident lytic bone lesions, which, at the highest dose, were undetectable. Histologically, OPG also decreased skeletal tumor burden and tumor-associated osteoclasts. In a nude mouse model, OPG treatment completely prevented radiographic osteolytic lesions caused by human MDA-MB-231 breast cancer cells. Histologically, OPG decreased skeletal tumor burden by 75% and completely eradicated MDA tumor-associated osteoclasts. In both models, OPG had no effect on metastatic tumor burden in a panel of soft tissue organs. These data indicate that OPG may be an effective therapy for preventing osteolysis and decreasing skeletal tumor burden in patients with bone metastasis.

Topics: Adenocarcinoma; Animals; Bone Neoplasms; Breast Neoplasms; Cell Line, Transformed; Colonic Neoplasms; Disease Models, Animal; Dose-Response Relationship, Drug; Glycoproteins; Humans; Mice; Mice, Inbred BALB C; Mice, Inbred DBA; Mice, Nude; Osteolysis; Osteoprotegerin; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Xenograft Model Antitumor Assays