osteoprotegerin and Multiple-Myeloma

Receptor activator of nuclear factor-kappa B (RANK) and its ligand, RANKL, are expressed in a variety of tissues throughout the body; their primary role is in the regulation of bone remodeling and development of the immune system. Consistent with these functions, evidence exists for a role of RANK/RANKL in all stages of tumorigenesis, from cell proliferation and carcinogenesis to epithelial-mesenchymal transition to neoangiogenesis and intravasation to metastasis to bone resorption and tumor growth in bone. Results from current studies also point to a role of RANK/RANKL signaling in patients with multiple myeloma, who have increased serum levels of soluble RANKL and an imbalance in RANKL and osteoprotegerin. Current therapies for patients with multiple myeloma demonstrate that RANKL may be released by tumor cells or osteoprogenitor cells. This article will review currently available evidence supporting a role for RANK/RANKL signaling in tumorigenesis, with a focus on patients with multiple myeloma.

Topics: Bone Remodeling; Carcinogenesis; Cell Proliferation; Epithelial-Mesenchymal Transition; Humans; Multiple Myeloma; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Signal Transduction

Osteolytic bone disease is a frequent complication of multiple myeloma, resulting in skeletal complications that are a significant cause of morbidity and mortality. It is the result of an increased activity of osteoclasts, which is not followed by reactive bone formation by osteoblasts. Recent studies have revealed novel molecules and pathways that are implicated in osteoclast activation and osteoblast inhibition. Among them, the most important include the receptor activator of nuclear factor-kappa B ligand/osteoprotegerin pathway, the macrophage inflammatory proteins and the activin-A that play a crucial role in osteoclast stimulation in myeloma, while the wingless-type (Wnt) signalling inhibitors (sclerostin and dickkopf-1) along with the growth factor independence-1 are considered the most important factors for the osteoblast dysfunction of myeloma patients. Finally, the role of osteocytes, which is the key cell for normal bone remodelling, has also revealed during the last years through their interaction with myeloma cells that leads to their apoptosis and the release of RANKL and sclerostin maintaining bone loss in these patients. This review focuses on the latest available data for the mechanisms of bone destruction in multiple myeloma.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis; Bone Morphogenetic Proteins; Bone Resorption; Genetic Markers; Humans; Intercellular Signaling Peptides and Proteins; Multiple Myeloma; Osteoclasts; Osteocytes; Osteogenesis; Osteolysis; Osteoprotegerin; RANK Ligand; Wnt Signaling Pathway

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

The presence of multiple myeloma (MM) in a patient with systemic sclerosis is a rare and unusual occurrence with unclear significance. We report the case of a 55-year-old woman with a 20-year history of systemic sclerosis, who subsequently presented with clinical stage IIIA IgG-λ MM. The systemic sclerosis was diagnosed and treated in 1988 with D: -penicillamine and methotrexate. Twenty years later, in December 2008, she presented with symptoms of Raynaud's phenomenon and intense facial pruritus. Immunoelectrophoresis confirmed the presence of a IgG-λ paraprotein (71.90 g/l) and Bence Jones proteinuria of the lambda light chains. Bone marrow histology revealed infiltrates of plasmocytes and lymphoplasmocytes which were on immunohistochemistry CD38+, FGF-R3+ and OPG+. An extensive X-ray skeletal survey did not show any osteolytic lesions or fractures. The patient was treated according to the CTD protocol (cyclophosphamide, thalidomide, and dexamethasone) which was effective against the myeloma as well as the systemic sclerosis and patient achieved complete remission.

Topics: Bone Marrow; Female; Humans; Middle Aged; Multiple Myeloma; Osteoprotegerin; Receptor, Fibroblast Growth Factor, Type 3; Scleroderma, Systemic

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

Healthy jawbones ensure better tooth anchorage and the ability to masticate and maintain metabolism. This is achieved by a delicate balance between bone formation and resorption in response to functional demands. An imbalance in the expression of receptor activator of nuclear factor kappa-B (RANK) ligand (RANKL) and osteoprotegerin (OPG) or osteoclastogenesis inhibitory factor (OCIF) is believed to be the underlying mechanism of osteolysis in metastases, multiple myelomas, and cancer therapy-induced bone loss in patients. Considered mainly as bone-specific agents to treat postmenopausal osteoporosis, bisphosphonates, in combination with certain chemotherapeutic agents have proved to be effective in prevention of tumor formation and metastatic osteolysis in bone tissue. Osteonecrosis of the jaws associated with them has, however, been of grave concern to the prosthodontist, as it predisposes patients to a bone-deficient basal seat for dental prostheses. This manuscript reviews available information over the past 13 years on possible mechanisms of bone loss, bisphosphonate-induced osteonecrosis of jaw bones, and prosthodontic concerns.

Topics: Alveolar Bone Loss; Bisphosphonate-Associated Osteonecrosis of the Jaw; Bone Density Conservation Agents; Bone Neoplasms; Contraindications; Dental Implants; Dental Prosthesis Retention; Diphosphonates; Humans; Multiple Myeloma; Osteoclasts; Osteolysis; Osteoprotegerin; Parathyroid Hormone-Related Protein; RANK Ligand

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

Osteoclastic bone resorption is a critical component of skeletal complications of malignancy including fracture, bone pain, hypercalcemia, and spinal cord compression. Three proteins, RANKL, RANK, and OPG have been recently identified as key determinants of osteoclastogenesis and the regulation of bone resorption. Both RANKL and OPG can be aberrantly regulated in the cancer setting and function as important gatekeepers of tumor-induced osteolytic bone disease. RANKL-induced osteoclastogenesis not only mediates osteolytic bone disease, but also contributes to the pathogenesis of osteoblastic bone disease resulting from tumors. In addition, an important role was recently described for bone marrow derived RANKL to mediate the bone-specific tropism of RANK-expressing tumor cells. This manuscript will review how RANKL contributes to skeletal complications of cancer and the development of targeted, mechanism-based drugs that inhibit RANKL.

Topics: Bone Diseases; Bone Neoplasms; Bone Resorption; Drug Delivery Systems; Humans; Models, Biological; Multiple Myeloma; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Signal Transduction

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

Hypercalcemia arises in the advanced stage of myeloma due to marked increase in osteoclastic bone resorption by local factors in the bone marrow, and is among the most serious complications in myeloma. It can progress rapidly and provoke acute renal failure and coma, which may become life-threatening. Early detection by serum calcium levels and treatment of hypercalcemia is important, although its clinical symptoms are difficult to distinguish from those in other underlying complications. Bisphosphonates, potent inhibitors of bone resorption, are recommended as the most effective therapy and have been approved for treatment of hypercalcemia in myeloma.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Biomarkers; Bone Density Conservation Agents; Bone Resorption; Calcium; Denosumab; Diphosphonates; Humans; Hypercalcemia; Multiple Myeloma; Osteoclasts; Osteoprotegerin; RANK Ligand

Multiple myeloma is characterized by accumulation of monoclonal plasma cells in the bone marrow and progression of lytic bone lesions. Myeloma cells enhance bone resorption by triggering a coordinated increase in RANK ligand and decrease in osteoprotegerin in the bone marrow. Macrophage inflammatory protein (MIP)-1alpha and MIP-1beta are secreted by myeloma cells, and play a major role in the enhancement of bone resorption by myeloma cells. Furthermore, the growth and survival of myeloma cells are enhanced by contact with osteoclasts, suggesting the presence of a vicious cycle between bone destruction and myeloma cell expansion. In addition, myeloma cells secrete soluble Wnt inhibitors, dickkopf (Dkk)-1 and secreted Frizzled-related protein (sFRP)-2, to suppress bone formation. Thus, myeloma cells closely interact with bone cells in the bone marrow microenvironment to enhance bone resorption and concomitantly suppress bone formation, which causes the formation of destructive bone lesions and a rapid loss of bone. Disruption of the pathognomonically skewed cellular interactions in myeloma bone marrow microenvironment may ameliorate bone destruction along with myeloma expansion.

Topics: Bone Diseases; Bone Resorption; Carrier Proteins; Cell Differentiation; Chemokine CCL3; Chemokine CCL4; Chemokines, CC; Glycoproteins; Humans; Macrophage Inflammatory Proteins; Membrane Glycoproteins; Multiple Myeloma; Osteoblasts; Osteoclasts; Osteogenesis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Wnt Proteins

The cells of the malignant clone of plasmacell myeloma have cytogenetic aberrations in a substantial number of cases. Many of these abnormal karyotypes are predictive for an unfavorable outcome. Gene mutations and abnormal gene expression, particularly of oncogenes and tumor suppressor genes, are often observed in myeloma cells. The cross talk between the myeloma cells and the bone marrow microenvironment plays an important role for growth and survival of the tumor cells. As a consequence of this cell-to-cell-interaction, several cytokines are secreted. The intracellular signaling, evoked by these cytokines, leads to continuous growth and proliferation and inhibition of apoptosis. Since these molecular pathways have been defined, many new targets for therapeutical interventions become obvious. Some molecules, directed against cytokines, are under early clinical investigation. Medicaments intervening in the cross talk between the myeloma cell and the bone marrow stroma as Thalidomide, Lenalidomide or Bortezomib are already available. Many of the myeloma patients suffer from bone disease. Some new drugs inhibiting the differentiation and activation of osteoclasts are evaluated in clinical trials. These molecules will be an important contribution against the painful bone disease of plasmacell myeloma.

Topics: Aged; Angiogenesis Inhibitors; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Arsenic Trioxide; Arsenicals; Boronic Acids; Bortezomib; Cell Communication; Chromosome Aberrations; Gene Expression; Glycoproteins; Growth Inhibitors; Humans; Immunologic Factors; Immunosuppressive Agents; Lenalidomide; Multiple Myeloma; Osteoporosis; Osteoprotegerin; Oxides; Protease Inhibitors; Pyrazines; Randomized Controlled Trials as Topic; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Thalidomide; Time Factors

Multiple myeloma (MM) almost exclusively develops in the bone marrow and generates devastating bone destruction by osteoclasts (OCs) recruited around myeloma cells. The severity of bone disease correlates with tumor burden. The interaction between RANK, expressed on the surface of OCs, and RANKL, a key molecule in the regulation of osteoclastogenesis expressed on bone marrow stromal cells, plays a role in the development and activation of OCs, whereas OPG, a decoy receptor for RANKL secreted from stromal cells, inhibits RANKL/RANK signaling. Myeloma cells stimulate osteoclastogenesis by triggering an increase in RANKL and decrease in OPG in bone marrow cells. They also express syndecan-1, a molecule which binds and stimulates OPG degradation in myeloma cells. MM cells can themselves express RANKL which can interact directly with RANK on OCs to promote osteoclast formation in a stromal cell-independent manner. Moreover, myeloma cells may additionally inhibit osteoblastogenesis directly or indirectly. The mechanisms involved in these coordinated processes are described and discussed.

Topics: Bone Resorption; Humans; Multiple Myeloma; Osteoclasts; Osteoprotegerin; RANK Ligand

Histomorphometry and biochemical markers of bone turnover have shown that, although osteoclast activity is increased in multiple myeloma (MM), mostly through the receptor activator of nuclear factor-kappaB ligand/osteoprotegerin axis, the key element in vivo to determine the presence or absence of osteolytic lesions resides on the presence and activity of osteoblasts. The loss of bone observed in MM is the result of an uncoupling of bone formation and bone resorption. Bortezomib is a first-in-class proteasome inhibitor developed as an antineoplastic agent with marked activity in relapsed/refractory MM. Response to bortezomib has been related to a significant increase in alkaline phosphatase (ALP). Increased ALP in patients responding to bortezomib was associated with a parallel increase in bone-specific ALP and parathyroid hormone, suggesting that response to bortezomib in MM is closely associated with osteoblastic activation. Variation in markers of osteoblastic activation (such as ALP) have also predicted response and response duration in patients with myeloma treated with bortezomib (P < 0.0001). This clinical observation has been confirmed in an experimental mouse model for primary human myeloma. The consequences of increased bone anabolism on myeloma growth need to be closely evaluated in prospective trials.

Topics: Alkaline Phosphatase; Biomarkers; Bone Remodeling; Boronic Acids; Bortezomib; Clinical Trials as Topic; Humans; Multiple Myeloma; NF-kappa B; Osteoblasts; Osteoclasts; Osteoprotegerin; Parathyroid Hormone; Prospective Studies; Protease Inhibitors; Proteasome Inhibitors; Pyrazines

Bone disease in patients with multiple myeloma (MM) is characterized by uncoupled bone remodeling, evident as enhanced osteolytic resorption and decreased rather than increased bone formation. MM-triggered osteolysis follows deregulation of the receptor activator of nuclear factor kappaB ligand (RANKL)/osteoprotegerin cytokine axis. Inhibition of bone formation may result from the ability of MM to inhibit the function of Wnts, secreted glycoproteins critical to osteoblast development. Recent studies show how these processes may be linked.

Topics: Animals; Bone Remodeling; Humans; Multiple Myeloma; Osteoprotegerin; RANK Ligand; Signal Transduction; Wnt Proteins

A new understanding of the regulation of bone resorption developed with the discovery of receptor activator of nuclear factor-kappaB, receptor activator of nuclear factor-kappaB ligand, and osteoprotegerin in 1997-1998. The RANK signaling system is abnormally regulated in multiple myeloma, and this favors increased osteoclast function, which early in the disease is compensated by increased osteoblast function. Later in the disease osteoblast activity decreases, resulting in osteolytic lesions. We review the factors implicated in osteoclast and osteoblast function. Among these are receptor activator of nuclear factor-kappaB, receptor activator of nuclear factor-kappaB ligand, osteoprotegerin, hepatocyte growth factor, macrophage inflammatory protein-1alpha, bone morphogenetic proteins, and the Wnt system. Bisphosphonates are the only drugs used in routine clinical management; however, the complex regulation system of bone homeostasis offers a number of of possible targets for therapy, which are discussed.

Topics: Bone and Bones; Bone Diseases; Cytokines; Diphosphonates; Humans; Multiple Myeloma; NF-kappa B; Osteoblasts; Osteoclasts; Osteoprotegerin; Receptor Activator of Nuclear Factor-kappa B

Topics: Anorexia Nervosa; Biomarkers; Cardiovascular Diseases; Diabetes Mellitus; Enzyme-Linked Immunosorbent Assay; Female; Glycoproteins; Humans; Kidney Diseases; Male; Multiple Myeloma; Osteitis Deformans; Osteoprotegerin; Prostatic Neoplasms; Reagent Kits, Diagnostic; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Reference Values; Specimen Handling; Urinary Bladder Neoplasms

One of the most characteristic features of multiple myeloma is the development of osteolytic bone lesions. Myeloma-associated bone disease is caused by an increase in osteoclastic bone resorption and a decrease in osteoblastic new bone formation. Insight into the molecular mechanisms of osteoclastogenesis has been provided by the detection of receptor activator of NF-kappaB ligand (RANKL), its specific receptor (RANK) and its decoy receptor antagonist osteoprotegerin (OPG). The RANK signaling system is abnormally regulated in multiple myeloma and targeting this system may ameliorate myeloma bone disease. Less is known about the development of osteoblastic dysfunction, and further knowledge about the interaction between myeloma cells and osteoblasts is required. The aim of this review is to focus on the principles of bone biology for a better understanding of the development of myeloma bone disease and to identify possible therapeutic targets.

Topics: Bone Remodeling; Bone Resorption; Carrier Proteins; Cell Differentiation; Diphosphonates; Glycoproteins; Humans; Membrane Glycoproteins; Multiple Myeloma; Osteoblasts; Osteoclasts; Osteolysis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

The discovery of the signalling system consisting of receptor activator of nuclear factor-KB ligand (RANKL), its receptor RANKL and its decoy receptor osteoprotegerin (OPG) has been a key step in the understanding of pathophysiology of the bone microenvironment and has provided pharmacological targets for new anti-resorptive drugs. The system is central to the interactions among bone, vascular and immune cells. OPG and a soluble form of RANKL (sRANKL) are present in the blood stream. The measurement of their concentrations offers insights into the regulatory mechanisms of the system and the possibility of using new markers in a number of diseases. Besides bone metabolic disorders, the list includes malignancies, rheumatic and cardiovascular diseases. However, apparent discrepancies in the outcome of studies point to a number of caveats: lack of control of pre-analytical and analytical variables; paucity of data on the metabolic removal of the molecules; the cross sectional design of most studies comparing patients with healthy subjects. Finally, circulating OPG and sRANKL derive from several sources, and their concentrations may be alterated by different coexisting pathological processes. In the absence of tissue-specific isoforms, diagnostic or prognostic significance may be suggested from data obtained in large cohorts, but is still of limited usefulness in single patients.

Topics: Biomarkers; Bone and Bones; Bone Diseases, Metabolic; Bone Neoplasms; Cardiovascular Diseases; Carrier Proteins; Cohort Studies; Glycoproteins; Humans; Membrane Glycoproteins; Multiple Myeloma; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Risk Factors; Signal Transduction

Multiple myeloma is associated with the development of a devastating bone disease mediated by increased osteoclastic activity. The ligand for receptor activator of nuclear factor-kappaB (RANKL) plays a critical role in normal osteoclast biology and is abnormally regulated in myeloma. Targeting this system with recombinant decoy receptor, osteoprotegerin, or soluble forms of the receptor activator of nuclear factor-kappaB is able to prevent myeloma bone disease in pre-clinical models. Intriguingly, inhibiting osteoclast formation and bone resorption, and altering the bone marrow microenvironment, results in an indirect anti-myeloma effect.

Topics: Animals; Bone Resorption; Carrier Proteins; Glycoproteins; Humans; Membrane Glycoproteins; Multiple Myeloma; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

The increase of osteoclast activation and formation is mainly involved in the development of the osteolytic bone lesions that characterize multiple myeloma (MM) patients. The mechanisms by which myeloma cells induce bone resorption have not been clear for many years. Recently, new evidence has elucidated which factors are critically involved in the activation of osteoclastic cells in MM. The potential role of the critical osteoclastogenic factor, the receptor activator of NF-kappaB ligand (RANKL), and its soluble antagonist osteoprotegerin (OPG) in the activation of bone resorption in MM is summarized in this review. It has been demonstrated that human MM cells induce an imbalance in the bone marrow environment of the RANKL/OPG ratio in favor of RANKL that triggers the osteoclast formation and activation leading to bone destruction. The direct production of the chemokine macrophage inflammatory protein-1 alpha (MIP-1alpha) by myeloma cells, in combination with the RANKL induction in BM stromal cells in response to myeloma cells, are critical in osteoclast activation and osteoclastogenesis.

Topics: Animals; Bone Resorption; Carrier Proteins; Chemokine CCL3; Chemokine CCL4; Disease Models, Animal; Glycoproteins; Humans; Interleukin-7; Macrophage Inflammatory Proteins; Membrane Glycoproteins; Multiple Myeloma; Osteoclasts; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Stromal Cells

The excessive bone resorption observed in multiple myeloma may be due to the production of several osteoclast-activating factors either by the myeloma cells themselves or by the bone marrow microenvironment. These factors could act primarily via a common final pathway involving the recently-described members of the TNF receptor-ligand family: RANKL (Receptor Activator of NK-kappaB Ligand) and its corresponding RANK receptor that play a crucial role in osteoclast differentiation and activation, and osteoprotegerin (OPG), the physiological inhibitor of RANKL. RANKL expression by stromal cells is increased in myeloma and is associated with a concomitant decrease in OPG expression. This increase in RANKL-OPG ratio correlates with the extent of the myeloma bone disease. The RANKL-OPG imbalance could play a decisive role in the lytic bone lesions in myeloma, and this possibility is reinforced by several in-vivo studies that have assessed the effects of administering RANKL inhibitors in murine myeloma models. Treatment with either OPG: Fc or RANK: Fc decreased myeloma osteolysis in these models. RANKL blockade is also currently being evaluated in malignant osteolysis in humans. A therapeutic approach targeting the RANKL-RANK signaling pathway could be of great value, as RANKL inhibitors are potent anti-resorptive agents, affecting both myeloma-induced bone resorption and the tumor burden.

Topics: Animals; Bone Resorption; Carrier Proteins; Glycoproteins; Humans; Immunotherapy; Ligands; Membrane Glycoproteins; Multiple Myeloma; NF-kappa B; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

The emergence of the molecular triad osteoprotegerin (OPG)/Receptor Activator of NF-kB (RANK)/RANK Ligand (RANKL) has helped elucidate a key signalling pathway between stromal cells and osteoclasts. The interaction between RANK and RANKL plays a critical role in promoting osteoclast differentiation and activation leading to bone resorption. OPG is a soluble decoy receptor for RANKL that blocks osteoclast formation by inhibiting RANKL binding to RANK. The OPG/RANK/RANKL system has been shown to be abnormally regulated in several malignant osteolytic pathologies such as multiple myeloma [MM, where enhanced RANKL expression (directly by tumour cells or indirectly by stromal bone cells or T-lymphocytes)] plays an important role in associated bone destruction. By contrast, production of its endogenous counteracting decoy receptor OPG is either inhibited or too low to compensate for the increase in RANKL production. Therefore, targeting the OPG/RANK/RANKL axis may offer a novel therapeutic approach to malignant osteolytic pathologies. In animal models, OPG or soluble RANK was shown both to control hypercalcaemia of malignancy and the establishment and progression of osteolytic metastases caused by various malignant tumours. To this day, only one phase I study has been performed using a recombinant OPG construct that suppressed bone resorption in patients with multiple myeloma or breast carcinoma with radiologically confirmed bone lesions. RANK-Fc also exhibits promising therapeutic effects, as revealed in animal models of prostate cancer and multiple myeloma. If the animal results translate to similar clinical benefits in humans, using RANK-Fc or OPG may yield novel and potent strategies for treating patients with established or imminent malignant bone diseases and where standard therapeutic regimens have failed.

Topics: Antineoplastic Agents; Bone Neoplasms; Carrier Proteins; Cell Transformation, Neoplastic; Glycoproteins; Humans; Membrane Glycoproteins; Multiple Myeloma; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Signal Transduction

Topics: Biomarkers; Bone Resorption; Carrier Proteins; Enzyme-Linked Immunosorbent Assay; Glycoproteins; Humans; Membrane Glycoproteins; Multiple Myeloma; Osteoclasts; Osteoporosis, Postmenopausal; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Solubility; Specimen Handling

Multiple myeloma (MM) is a plasma cell malignancy characterized by the high capacity to induce osteolytic bone lesions that mainly result from an increased bone resorption related to the stimulation of osteoclast recruitment and activity. Although it is known that myeloma cells induce osteoclastic bone resorption, the biological mechanisms involved in the pathophysiology of MM-induced bone resorption have been unclear for several years. Recently, new data seem to elucidate which mechanism is critically involved in the activation of osteoclastic cells in MM. The critical osteoclastogenetic factor RANKL and its soluble antagonist osteoprotegerin (OPG) are the major candidates in the pathophysiology of MM bone disease. Human MM cells induce an imbalance in the RANKL/OPG ratio in the bone marrow environment that triggers the osteoclast formation and activation leading to bone destruction. The role or RANKL/OPG system and other osteoclast stimulating factors in the pathophysiology of MM bone disease are summarized in this update.

Topics: Animals; Bone Marrow; Bone Resorption; Carrier Proteins; Cell Differentiation; Cell Line, Tumor; Chemokine CCL4; Culture Media, Conditioned; Cytokines; Glycoproteins; Humans; Interleukin-6; Interleukin-7; Macrophage Inflammatory Proteins; Membrane Glycoproteins; Mice; Multiple Myeloma; Neoplasm Proteins; Osteoblasts; Osteoclasts; Osteolysis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Stromal Cells; T-Lymphocyte Subsets

Myeloma bone disease is due to interactions of myeloma cells with the bone marrow microenvironment, and is associated with pathologic fractures, neurologic symptoms and hypercalcemia. Adjacent to myeloma cells, the formation and activation of osteoclasts is increased, which results in enhanced bone resorption. The recent characterization of the essential cytokine of osteoclast cell biology, receptor activator of NF-kappa B ligand (RANKL) and its antagonist osteoprotegerin (OPG), have led to a detailed molecular and cellular understanding of myeloma bone disease. Myeloma cells induce RANKL expression in bone marrow stromal cells, and direct RANKL expression by myeloma cells may contribute to enhanced osteoclastogenesis in the bone microenvironment in myeloma bone disease. Furthermore, myeloma cells inhibit production and induce degradation of OPG. These effects result in an increased RANKL-to-OPG ratio that favors the formation and activation of osteoclasts. Patients with myeloma bone disease have inappropriately low serum and bone marrow levels of OPG. Specific blockade of RANKL prevented the skeletal complications in various animal models of myeloma, and suppressed bone resorption in a preliminary study of patients with myeloma bone disease.

Topics: Animals; Bone Neoplasms; Carrier Proteins; Gene Expression; Glycoproteins; Humans; Membrane Glycoproteins; Multiple Myeloma; Osteoblasts; Osteoclasts; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Severe bone destruction due to inappropriate osteoclastogenesis is a prominent feature of multiple myeloma (MM). MM increases bone loss by disrupting the checks that normally control signaling by receptor activator of nuclear factor kappaB ligand (RANK-L, also called TRANCE [tumor necrosis factor-related, activation-induced cytokine], osteoprotegerin ligand [OPG-L], osteoclast differentiation factor [ODF], and tumor necrosis factor superfamily member 11 [TNFSF11]), a TNF-family cytokine required for osteoclast differentiation and activation. RANK-L binds to its functional receptor RANK (TNF receptor superfamily member 11a [TNF RSF11a]) to stimulate osteoclastogenesis. Osteotropic cytokines regulate this process by controlling bone marrow stromal expression of RANK-L. Further control over osteoclastogenesis is maintained by regulated expression of osteoprotegerin (OPG, also called osteoclastogenesis inhibitory factor and TNFRSF11b), a soluble decoy receptor for RANK-L. In normal bone marrow, abundant stores of OPG in stroma, megakaryocytes, and myeloid cells provide a natural buffer against increased RANK-L. MM disrupts these controls by increasing expression of RANK-L and decreasing expression of OPG. Concurrent deregulation of RANK-L and OPG expression is found in bone marrow biopsies from patients with MM but not in specimens from patients with non-MM hematologic malignancies.. RANK-Fc is a recombinant RANK-L antagonist that is formed by fusing the extracellular domain of RANK to the Fc portion of human immunoglobulin G(1) (hIgG(1)). In vitro, addition of RANK-Fc virtually eliminates the formation of osteoclasts in cocultures of MM with bone marrow and osteoblast/stromal cells. The severe combined immunodeficiency (SCID)/ARH77 mouse model and the SCID-hu-MM mouse model of human MM were used to assess the ability of RANK-Fc to block the development of MM-induced bone disease in vivo. Mice received either RANK-Fc or hIgG(1) 200 microg intravenously three times per week.. RANK-Fc limited bone destruction in both the SCID/ARH-77 model and the SCID-hu-MM model. Administration of RANK-Fc also caused a marked reduction in tumor burden and serum paraprotein in SCID-hu-MM mice that was associated with the restoration of OPG and a reduction in RANK-L expression in the xenograft.. MM-induced bone destruction requires increased RANK-L expression and is facilitated by a concurrent reduction in OPG, a natural decoy receptor for RANK-L. Administration of the RANK-L antagonist RANK-Fc limits MM-induced osteoclastogenesis, development of bone disease, and MM tumor progression.

Topics: Animals; Carrier Proteins; Disease Models, Animal; Disease Progression; Glycoproteins; Humans; Immunoglobulin Fc Fragments; Immunoglobulin G; Membrane Glycoproteins; Mice; Mice, SCID; Multiple Myeloma; Osteoclasts; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Multiple myeloma (MM) is a plasma cell malignancy localized in the bone marrow (BM) and characterized by a high capacity for bone destruction. Almost all patients with MM have early osteolytic lesions, which result mainly from increased bone resorption related to stimulation of osteoclast recruitment and activity in the immediate vicinity of myeloma cells. The recent discovery of Osteoprotegerin (OPG) and the subsequent identification of its ligand RANKL have provided new insights in the regulation of osteoclastogenesis. The ratio OPG/RANKL is critical for the regulation of bone remodeling maintaining the balance between osteoblastic and osteoclastic activity. This review summarizes the new concept that myeloma cells induce in bone environment an imbalance in the OPG/RANKL system responsible for osteolysis observed in patients. Indeed, myeloma cells increase in bone environment the expression of the potent osteoclastogenic factor RANKL and decrease the osteoprotective factor OPG production. Biological mechanisms involved in these processes are discussed. Furthermore, the chemokines MIP-1alpha and MIP-1beta belonging to the RANTES family are potent osteoclastogenic factors produced by myeloma cells and participate in myeloma-associated bone disease. These data open new avenues for the treatment of bone disease in MM and highlight the promising therapeutical interest of RANKL inhibitors (OPG and RANK-Fc) and MIP-1 inhibitors in the management of myeloma-associated osteolysis, besides bisphosphonates.

Topics: Animals; Bone Remodeling; Carrier Proteins; Chemokine CCL3; Chemokine CCL4; Diphosphonates; Drug Design; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Hypercalcemia; Macrophage Inflammatory Proteins; Membrane Glycoproteins; Mice; Multiple Myeloma; Osteoclasts; Osteolysis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Recombinant Fusion Proteins

Topics: Bone Diseases; Bone Resorption; Carrier Proteins; Chemokine CCL4; Cytokines; Diphosphonates; Glycoproteins; Humans; Macrophage Inflammatory Proteins; Membrane Glycoproteins; Multiple Myeloma; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

The major clinical manifestations of multiple myeloma are related to the loss of bone. This bone loss often leads to pathologic fractures, spinal cord compression, hypercalcemia, and bone pain. This article reviews the cytokine network involved in myeloma bone disease; describes the signaling cascade involved in osteoclastogenesis and mechanisms of action of novel therapeutic options for myeloma bone disease such as osteoprotegerin, RANK human immunoglobulin fusion protein, the proteasome inhibitor PS-341, and bisphosphonates; and summarizes the latest clinical trial results using oral and intravenous bisphosphonates for bone disease in multiple myeloma.

Topics: Bone Resorption; Boronic Acids; Bortezomib; Carrier Proteins; Clinical Trials as Topic; Cytokines; Diphosphonates; Endothelial Growth Factors; Glycoproteins; Humans; Imidazoles; Immunoglobulin Fc Fragments; Intercellular Signaling Peptides and Proteins; Lymphokines; Membrane Glycoproteins; Multiple Myeloma; Osteoprotegerin; Pamidronate; Pyrazines; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Recombinant Fusion Proteins; Signal Transduction; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors; Zoledronic Acid

Topics: Antineoplastic Agents; Carrier Proteins; Cytokines; Diphosphonates; Glycoproteins; Humans; Membrane Glycoproteins; Multiple Myeloma; Osteoprotegerin; Pamidronate; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Osteolytic bone disease in multiple myeloma (MM) is caused by enhanced osteoclast (OCL) activation and inhibition of osteoblast function. Lenalidomide and bortezomib have shown promising response rates in relapsed and newly diagnosed MM, and bortezomib has recently been reported to inhibit OCLs. We here investigated the effect of lenalidomide on OCL formation and osteoclastogenesis in comparison with bortezomib. Both drugs decreased alpha V beta 3-integrin, tartrate-resistant acid phosphatase-positive cells and bone resorption on dentin disks. In addition, both agents decreased receptor activator of nuclear factor-kappaB ligand (RANKL) secretion of bone marrow stromal cells (BMSCs) derived from MM patients. We identified PU.1 and pERK as major targets of lenalidomide, and nuclear factor of activated T cells of bortezomib, resulting in inhibition of osteoclastogenesis. Furthermore, downregulation of cathepsin K, essential for resorption of the bone collagen matrix, was observed. We demonstrated a significant decrease of growth and survival factors including macrophage inflammatory protein-alpha, B-cell activating factor and a proliferation-inducing ligand. Importantly, in serum from MM patients treated with lenalidomide, the essential bone-remodeling factor RANKL, as well as the RANKL/OPG ratio, were significantly reduced, whereas osteoprotegerin (OPG) was increased. We conclude that both agents specifically target key factors in osteoclastogenesis, and could directly affect the MM-OCL-BMSCs activation loop in osteolytic bone disease.

Topics: Antineoplastic Agents; B-Cell Activating Factor; Bone Remodeling; Bone Resorption; Boronic Acids; Bortezomib; Cathepsin K; Cathepsins; Cells, Cultured; Chemokine CCL3; Humans; Integrin alphaVbeta3; Lenalidomide; Multiple Myeloma; Osteoclasts; Osteoprotegerin; Proto-Oncogene Proteins; Pyrazines; RANK Ligand; Thalidomide; Trans-Activators; Transcription Factor AP-1; Tumor Necrosis Factor Ligand Superfamily Member 13

The aim of this study was the evaluation of the effect of intermediate doses of thalidomide with dexamethasone (Thal/Dex) on disease course and bone disease in patients with refractory/relapsed myeloma who were under zoledronic acid therapy. We studied 35 patients, who received thalidomide at a dose of 200 mg/daily. We measured, pre-, 3 and 6 months post-treatment soluble receptor activator of nuclear factor-kappaB ligand (sRANKL), osteoprotegerin (OPG), osteopontin (OPN), markers of bone resorption and formation. Before treatment, patients had increased levels of sRANKL/OPG ratio, bone resorption markers and OPN, while they had suppressed bone formation. The pretreatment sRANKL/OPG ratio correlated with the extent of bone disease. Thal/Dex administration resulted in a significant reduction of sRANKL/OPG ratio, and bone resorption. Bone formation, OPG and OPN did not show any alteration. Changes of sRANKL/OPG ratio correlated with changes of bone resorption markers. Thal/Dex was given for a median time of 10 months and the median follow-up period was 22 months. The response rate was 65.7%. The median survival was 19.5 months. beta2-microglobulin, type of response and International Staging System predicted for survival. These results suggest that the combination of intermediate dose of Thal/Dex is effective in patients with refractory/relapsed myeloma and improves abnormal bone remodeling through the reduction of sRANKL/OPG ratio.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Biomarkers; Bone Remodeling; Dexamethasone; Dose-Response Relationship, Drug; Female; Glycoproteins; Humans; Ligands; Male; Middle Aged; Multiple Myeloma; Osteopontin; Osteoprotegerin; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Sialoglycoproteins; Survival Analysis; Thalidomide

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

BACKGROUND The aim of this study was to determine the effects of myeloma cells exposed to fluid shear stress on osteocytes and osteoclasts, and clarify the potential underlying mechanisms. MATERIAL AND METHODS A flow and a non-flow model were established using a flow fluid chamber. The myeloma cell line U266 and murine osteocytic MLO-Y4 cells were cultured in vitro. The osteocytes and osteoclasts were examined under a microscope. Osteoclasts were stained for tartrate-resistant acid phosphatase (TRAP) activity. RANKL and osteoprotegerin (OPG) gene expression were detected using reverse transcription-quantitative polymerase chain reaction. RESULTS Compared with the controls, Y4 cells cultured with U266 culture supernatant showed altered morphology, fewer osteocytes, increased RANKL gene expression, a higher RANKL/OPG gene ratio, and a greater number of TRAP-positive osteoclasts (P<0.05 for all). Compared to the no-flow model, the flow model showed a higher number of Y4 cells, increased OPG gene expression, decreased RANKL gene expression, a lower RANKL/OPG gene ratio, and fewer TRAP-positive osteoclasts (P<0.05 for all). CONCLUSIONS Our study revealed that fluid shear stress ameliorated the inhibitory effects of myeloma cells on osteocyte growth and inhibited osteoclast proliferation by means of decreasing RANKL/OPG gene expression. This may have clinical implications in patients with multiple myeloma in that mechanical loading with low-intensity vibration or mild exercise may prevent the progression of myeloma bone disease.

Topics: Animals; Biomechanical Phenomena; Cell Line, Tumor; Gene Expression Regulation; Humans; Hydrodynamics; Mice; Multiple Myeloma; Osteoclasts; Osteocytes; Osteoprotegerin; Receptor Activator of Nuclear Factor-kappa B; Stress, Mechanical; Tumor Microenvironment

Objective To observe the effect of curcumol on the biological behavior of multiple myeloma (MM) cells, thus studying its possible mechanisms for MM treatment. Methods Bone marrow mesenchymal stem cells (BMSCs) and multiple myeloma cell line 8226 (RPMI 8226) were taken as sub- jects, which were then divided into the RMPI 8226 group (cultured by RMPI 8226 alone) and the BMSCs + RMPI 8226 group (cultured by BMSCs and RMPI 8226). Curcumol in different concentrations (0. 1, 0.5, 1. 0 , 10.0 μg/mL) was added to cells in the two groups respectively. Cell proliferation, cell cycle, and ap' optosis induced by curcumol were examined by flow cytometry. The expressions of receptor activator of nuclear factor Kb ligand ( RANKL ) and osteoprotegerin ( OPG) were detected using reverse tran- scriptase-polymerase chain reaction (RT-PCR). Results Curcumol induced arrested cell cycle of RMPI 8226. The arrest of RMPI 8226 cell cycle was more obviously in the RMPI 8226 group than in the BMSCs + RMPI 8226 group. After curcumol treatment the cell proliferation of RPMI 8226 was significantly inhibited (P <0. 01) and its apoptosis was increased (P <0. 01). Co-cultured with BMSCs decreased curcumol in- duced apoptosis of RPMI 8226. Curcumol down-regulated the expression of osteogenic differentiation related gene RANKL, and up-regulated the expression of OPG. Conclusions Curcumol disturbed the cell cycle and induced apoptosis of RPMI 8226 cells. Curcumol up-regulated the expression of OPG as well as down-regulated the expression of RANKL. Co-culture with BMSCs could obviously inhibit curcumol in- duced apoptosis of MM cells, which might be associated with drug resistance of MM cells.

Topics: Bone Marrow Cells; Humans; Mesenchymal Stem Cells; Multiple Myeloma; Osteogenesis; Osteoprotegerin; RANK Ligand; Sesquiterpenes; Up-Regulation

The relationship between bone marrow (BM) cytokine and chemokine levels, cytogenetic profiles and skeletal involvement in multiple myeloma (MM) patients is not yet defined. This study investigated a cohort of 455 patients including monoclonal gammopathy of uncertain significance (MGUS), smoldering MM and symptomatic MM patients. Skeletal surveys, positron emission tomography (PET)/computerized tomography (CT) and magnetic resonance imaging (MRI) were used to identify myeloma bone disease. Significantly higher median BM levels of both C-C motif Ligand (CCL)3 and CCL20 were found in MM patients with radiographic evidence of osteolytic lesions as compared with those without, and in all MM patients with positive PET/CT scans. BM levels of CCL3, CCL20, Activin-A and Dickkopf-1 (DKK-1) were significantly higher in patients with high bone disease as compared with patients with low bone disease. Moreover, CCL20 BM levels were significant predictors of osteolysis on X-rays by multivariate logistic analysis. On the other hand, DKK-1 levels were related to the presence of MRI lesions independently of the osteolysis at the X-rays. Our data define the relationship between bone disease and the BM cytokine and chemokine patterns highlighting the tight relationship between CCL20 BM levels and osteolysis in MM.

Topics: Aged; Bone Marrow; Chemokine CCL20; Chemokine CCL3; Chemokines; Chromosome Aberrations; Cytokines; Female; Humans; Intercellular Signaling Peptides and Proteins; Magnetic Resonance Imaging; Male; Middle Aged; Multiple Myeloma; Osteolysis; Osteoprotegerin; RANK Ligand

A major complication of multiple myeloma (MM) is the development of osteolytic lesions, fractures and bone pain. To identify genetic variants influencing the development of MM bone disease (MBD), we analyzed MM patients of European ancestry (totaling 3774), which had been radiologically surveyed for MBD. Each patient had been genotyped for ~6 00 000 single-nucleotide polymorphisms with genotypes for six million common variants imputed using 1000 Genomes Project and UK10K as reference. We identified a locus at 8q24.12 for MBD (rs4407910, OPG/TNFRSF11B, odds ratio=1.38, P=4.09 × 10(-9)) and a promising association at 19q13.43 (rs74676832, odds ratio=1.97, P=9.33 × 10(-7)). Our findings demonstrate that germline variation influences MBD and highlights the importance of RANK/RANKL/OPG pathway in MBD development. These findings will contribute to the development of future strategies for prevention of MBD in the early precancerous phases of MM.

Topics: Aged; Biomarkers, Tumor; Bone Diseases; Female; Genotype; High-Throughput Nucleotide Sequencing; Humans; Male; Middle Aged; Multiple Myeloma; Neoplasm Staging; Osteoprotegerin; Polymorphism, Single Nucleotide; Prognosis; Risk Factors

miR-21 is an oncogenic microRNA (miRNA) with an emerging role as therapeutic target in human malignancies, including multiple myeloma (MM). Here we investigated whether miR-21 is involved in MM-related bone disease (BD). We found that miR-21 expression is dramatically enhanced, while osteoprotegerin (OPG) is strongly reduced, in bone marrow stromal cells (BMSCs) adherent to MM cells. On this basis, we validated the 3'UTR of OPG mRNA as miR-21 target. Constitutive miR-21 inhibition in lentiviral-transduced BMSCs adherent to MM cells restored OPG expression and secretion. Interestingly, miR-21 inhibition reduced RANKL production by BMSCs. Overexpression of protein inhibitor of activated STAT3 (PIAS3), which is a direct and validated target of miR-21, antagonized STAT3-mediated RANKL gene activation. Finally, we demonstrate that constitutive expression of miR-21 inhibitors in BMSCs restores RANKL/OPG balance and dramatically impairs the resorbing activity of mature osteoclasts. Taken together, our data provide proof-of-concept that miR-21 overexpression within MM-microenviroment plays a crucial role in bone resorption/apposition balance, supporting the design of innovative miR-21 inhibition-based strategies for MM-related BD.

Topics: 3' Untranslated Regions; Bone Marrow Cells; Bone Resorption; Cell Adhesion; Cell Line, Tumor; Coculture Techniques; HEK293 Cells; Humans; Interleukin-6; Lentivirus; Mesenchymal Stem Cells; MicroRNAs; Molecular Chaperones; Multiple Myeloma; Osteoclasts; Osteoprotegerin; Protein Inhibitors of Activated STAT; RANK Ligand; RNA, Messenger; STAT3 Transcription Factor; Stromal Cells; Tumor Microenvironment; Up-Regulation

Bone disease is one of the hallmarks of multiple myeloma (MM). The role of osteoprotegerin (OPG) in the RANK/RANKL/OPG signaling system is well defined in the myeloma bone disease. Polymorphisms of the TNFRSF11B gene encoding OPG have been studied in various bone diseases. However, relationship between the levels of OPG and development of bone lesions regardless of RANKL is yet unknown. In this study, the effects of OPG gene polymorphism on the development of bone lesions in MM were investigated.. C950T and C1181G polymorphisms of the OPG gene were studied in 52 MM patients (36 with bone lesions and 16 without bone lesions) and in another 20 control subjects using DNA sequencing.. 1181 G and 950 T alleles were overrepresented in MM patients having bone lesions. 950 TT/1181 GG haplotype frequency and TT/GG combined haplotype were also higher in MM patients having bone lesions compared to MM patients without bone lesions or to control.. This is the first study searching for the relationship between OPG gene variants C950T (promoter), C1181G (exon 1), and myeloma bone disease. It was concluded that the presence of polymorphic 1181 G/950 T alleles and 950 TT/1181 GG genotypes may play a role in the development of bone disease.

Topics: Aged; Bone Diseases; Female; Gene Frequency; Genotype; Humans; Male; Middle Aged; Multiple Myeloma; Osteoprotegerin; Polymorphism, Single Nucleotide

The bone marrow microenvironment provides important signals for the survival and proliferation of hematopoietic and malignant cells. In multiple myeloma, plasma cells are surrounded by stromal cells including osteoblasts. These stromal cells protect multiple myeloma cells from apoptosis induced by chemotherapeutic agents. Osteoprotegerin (OPG), a soluble receptor of the cytokine TNF-related apoptosis-inducing ligand (TRAIL), is secreted by osteoblasts and has been implicated in the prevention of cell death induced by TRAIL in malignant cells. Previously, we have designed death receptor-specific TRAIL variants that induce apoptosis exclusively via one of its death receptors. Here, we have studied in detail the interaction between recombinant human (rhTRAIL) variants and OPG. We show that a DR5-specific variant (rhTRAIL D269H/E195R) displays a significantly decreased affinity to OPG. Furthermore, this rhTRAIL variant shows a much higher activity when compared with rhTRAIL WT and retains its effectiveness in inducing cell death in multiple myeloma cell lines, in the presence of OPG secreted by stromal cells. We also demonstrate that stromal cells are largely insensitive to high concentrations of this rhTRAIL variant. In conclusion, rhTRAIL D269H/E195R is a potential therapy for multiple myeloma due to its high effectiveness and diminished binding to OPG.

Topics: Amino Acid Substitution; Apoptosis; Binding, Competitive; Blotting, Western; Bone Marrow; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Humans; Multiple Myeloma; Mutant Proteins; Mutation; Osteoprotegerin; Protein Binding; Recombinant Proteins; Surface Plasmon Resonance; TNF-Related Apoptosis-Inducing Ligand; Tumor Microenvironment

Bone metastases are frequently observed in patients with certain types of cancer and are significant cause of morbidity. Zoledronic acid (ZA) is routinely prescribed for patients with bone metastases by affecting osteoclast function. We aimed to assess the effect of ZA over time in patients with bone metastases by analyzing novel bone turnover marker levels including receptor activator of nuclear factor-k B ligand (RANKL) and osteoprotegerin (OPG) in serum and gingival crevicular fluid (GCF). Also, associations between these bone turnover markers with hematological and biochemistry dysregulation were studied. The study enrolled patients with bone metastases including 32 patients diagnosed with solid tumors and 15 patients with multiple myeloma. In these patients, GCF and serum RANKL and OPG levels were measured and compared with measures of hematological and biochemical parameters before and after 3 months of ZA therapy. Mean subject age was 54 years old with a range of 28-80 years. Skeletal-related events were observed in 8.5% of all patients. After the 3-month treatment of ZA therapy, no significant differences were found in serum and GCF levels of RANKL and OPG when compared with before treatment levels. GCF RANKL levels at baseline and following 3 months of ZA therapy were significantly higher in patients with solid tumors when compared patients diagnosed with multiple myeloma (p=0.001; p<0.001, respectively). GCF OPG levels after the entire course of ZA therapy were greater in patients with 5 or more bone metastases (p=0.04). For patients with multiple myeloma, control GCF OPG was negatively correlated with control platelet and WBC counts (p=0.018 and p=0.027, respectively). A negative correlation was observed between control serum RANKL and control serum OPG levels in myeloma patients (p=0.001). After 3 months of ZA therapy, no significant differences were observed in GCF and serum RANKL and OPG levels when compared with baseline. A negative correlation was observed between serum control RANKL and OPG levels in myeloma patients. OPG levels were greater in patients with 5 or more bone metastases. In patients diagnosed with multiple myeloma, GCF OPG levels were negatively associated with WBC and platelet counts.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers; Bone Density Conservation Agents; Bone Neoplasms; Diphosphonates; Female; Follow-Up Studies; Gingival Crevicular Fluid; Humans; Imidazoles; Male; Middle Aged; Multiple Myeloma; Neoplasm Staging; Neoplasms; Osteoprotegerin; Prognosis; RANK Ligand; Survival Rate; Zoledronic Acid

Osteolytic bone diseases are a prominent feature of multiple myeloma (MM), resulting from aberrant osteoclastic bone resorption that is uncoupled from osteoblastic bone formation. Myeloma stimulates osteoclastogenesis, which is largely dependent on an increase in receptor activator of NF-κB ligand (RANKL) and a decrease in osteoprotegerin (OPG) within the bone marrow milieu. Recently, brain-derived neurotrophic factor (BDNF) was identified as a MM-derived factor that correlates with increased RANKL levels and contributes to osteolytic bone destruction in myeloma patients. Because tyrosine receptor kinase B (TrkB), the receptor of BDNF, is abundantly expressed in osteoblasts, we sought to evaluate the role of BDNF/TrkB in myeloma-osteoblast interactions and the effect of this pathway on the RANKL/OPG ratio and osteoclastogenesis. Coculture systems constructed with noncontact transwells revealed that, in vitro, MM-derived BDNF increased RANKL and decreased OPG production in osteoblasts in a time- and dose-dependent manner. These effects were completely abolished by a specific small interfering RNA for TrkB. BDNF regulates RANKL/OPG expression in osteoblasts through the TrkB/ERK pathway. To investigate the biological effects of BDNF on myeloma in vivo, a SCID-RPMI8226 mice model was constructed using lentiviral short hairpin RNA-transfected RPMI8226 cells. In this system, stable knockdown of BDNF in MM cells significantly restored the RANKL/OPG homostasis, inhibited osteolytic bone destruction and reduced angiogenesis and tumor burden. Our studies provide further support for the potential osteoclastogenic effects of BDNF, which mediates stroma-myeloma interactions to disrupt the balance of RANKL/OPG expression, ultimately increasing osteoclastogenesis in MM.

Topics: Animals; Brain-Derived Neurotrophic Factor; Cell Line, Tumor; Female; Gene Silencing; Humans; MAP Kinase Signaling System; Mice; Multiple Myeloma; Osteoclasts; Osteolysis; Osteoprotegerin; RANK Ligand; Receptor, trkB; RNA, Small Interfering; Tumor Burden

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

Multiple myeloma (MM)-induced osteoclast (OC) formation is mainly due to an imbalance of the receptor activator NF-κB ligand (RANKL)-osteoprotegerin (OPG) ratio in favor of RANKL in the bone microenvironment and to the CCL3 production by MM cells. The purpose of the study was to investigate the effect of the immunomodulatory drugs on RANKL/OPG ratio, the production of pro-osteoclastogenic cytokines, and MM-induced OC formation. We found that in vivo concentrations of both lenalidomide (LEN) and pomalidomide (POM) significantly blunted RANKL upregulation normalizing the RANKL/OPG ratio in human osteoprogenitor cells (PreOBs) when co-cultured with MM cells and also inhibited CCL3 production by MM cells. A reduction in CD49d expression, a molecule critically involved in RANKL upregulation in the MM microenvironment, accompanied this effect. Consistently, the pro-osteoclastogenic property of MM cells co-cultured with PreOBs was reduced by both LEN and POM. We further investigated the effect of these drugs on the transcriptional profile of both MM cells and PreOBs by microarray analysis, which showed that adhesion molecules, such as ITGA8 and ICAM2, are significantly downregulated in MM cells. Our data suggest that LEN and POM inhibit MM-induced OC formation through normalization of the RANKL/OPG ratio targeting the expression of adhesion molecules by MM cells.

Topics: Antineoplastic Agents; Cell Adhesion Molecules; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cells, Cultured; Chemokine CCL3; Coculture Techniques; Dose-Response Relationship, Drug; Flow Cytometry; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunologic Factors; Integrin alpha4; Lenalidomide; Multiple Myeloma; Osteoblasts; Osteoprotegerin; RANK Ligand; Reverse Transcriptase Polymerase Chain Reaction; Thalidomide; Tumor Cells, Cultured; Tumor Microenvironment

The aim of this study was to explore the effects of brucine on bone metabolism in multiple myeloma (MM) and to compare brucine and bortezomib regarding the effects on MM in vitro. The half maximal inhibitory concentration (IC50) values of brucine and bortezomib in the MM cell line U266 were detected by MTT assay. In addition, the expression of alkaline phosphatase (ALP), osteocalcin (OC), osteoprotegerin (OPG) and osteoprotegerin ligand (also termed receptor activator of nuclear factor κB ligand) (RANKL) at mRNA levels were measured by RT-PCR. IC50 of bortezomib in the U266 cell line at 48 h was 22.4 nmol/l, and that of brucine was 0.16 nmol/l. Compared with osteoblasts incubated with MM cell supernatant alone, the mRNA levels of ALP, OC and OPG in osteoblasts co-treated with brucine and MM cell supernatant were higher (p<0.05), while the mRNA expression of RANKL was lower, and the ranges of the changes were all larger than those of the group treated with bortezomib (P<0.05). Brucine exerts effects on bone metabolism in multiple myeloma through the regulation of osteoclasts by osteoblasts.

Topics: Alkaline Phosphatase; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Bone and Bones; Boronic Acids; Bortezomib; Cell Differentiation; Cell Line, Tumor; Culture Media; Humans; Inhibitory Concentration 50; Multiple Myeloma; Osteoblasts; Osteocalcin; Osteoclasts; Osteoprotegerin; Polymerase Chain Reaction; Pyrazines; RANK Ligand; RNA, Messenger; RNA, Neoplasm; Stem Cells; Strychnine

Patients with multiple myeloma commonly develop focal osteolytic bone disease, as well as generalised osteoporosis. The mechanisms underlying the development of osteoporosis in patients with myeloma are poorly understood. Although disruption of the RANKL/OPG pathway has been shown to underlie formation of focal osteolytic lesions, its role in the development of osteoporosis in myeloma remains unclear. Increased soluble RANKL in serum from patients with myeloma raises the possibility that this molecule plays a key role. The aim of the present study was to establish whether sRANKL produced by myeloma cells contributes directly to osteoporosis. C57BL/KaLwRij mice were injected with either 5T2MM or 5T33MM murine myeloma cells. 5T2MM-bearing mice developed osteolytic bone lesions (p<0.05) with increased osteoclast surface (p<0.01) and reduced trabecular bone volume (p<0.05). Bone volume was also reduced at sites where 5T2MM cells were not present (p<0.05). In 5T2MM-bearing mice soluble mRANKL was increased (p<0.05), whereas OPG was not altered. In contrast, 5T33MM-bearing mice had no changes in osteoclast surface or trabecular bone volume and did not develop osteolytic lesions. Soluble mRANKL was undetectable in serum from 5T33MM-bearing mice. In separate experiments, RPMI-8226 human myeloma cells were transduced with an human RANKL/eGFP construct, or eGFP alone. RPMI-8226/hRANKL/eGFP cells, but not RPMI-8226/eGFP cells, stimulated osteoclastic bone resorption (p<0.05) in vitro. Sub-cutaneous injection of NOD/SCID mice with RPMI-8226/hRANKL/eGFP or RPMI-8226/eGFP cells resulted in tumour development in all mice. RPMI-8226/hRANKL/eGFP-bearing mice exhibited increased serum soluble hRANKL (p<0.05) and a three-fold increase in osteoclast number (p<0.05) compared to RPMI-8226/eGFP-bearing mice. This was associated with reduced trabecular bone volume (27%, p<0.05), decreased trabecular number (29%, p<0.05) and increased trabecular thickness (8%, p<0.05). Our findings demonstrate that soluble RANKL produced by myeloma cells causes generalised bone loss, suggesting that targeting RANKL may prevent osteoporosis in patients with myeloma.

Topics: Animals; Bone and Bones; Cell Line, Tumor; DNA, Complementary; Green Fluorescent Proteins; Humans; Lumbar Vertebrae; Mice; Mice, Inbred C57BL; Mice, SCID; Multiple Myeloma; Neoplasm Transplantation; Osteoclasts; Osteoporosis; Osteoprotegerin; RANK Ligand

Bisphosphonate induced osteonecrosis of the jaw (BONJ) is a complication in patients taking bisphosphonate (BP) that affects their quality of life and compliance. In this cohort study, patients with multiple myeloma (MM) on intravenous BP therapy were enrolled over 1 year. Demographic and clinical data and genotyping of 10 single nucleotide polymorphisms (SNPs) from seven candidate genes associated with drug or bone metabolism were determined. Of the 78 patients enrolled, 12 had BONJ. The median time to developing BONJ was 28 months. Univariate and multivariate analysis revealed a significant association between BONJ and smoking (p=0.048) and type of BP treatment (p=0.03). A trend for higher odds for BONJ was found for SNPs in five genes: COL1A1 (rs1800012), RANK (rs12458117), MMP2 (rs243865), OPG (rs2073618) and OPN (rs11730582). Considering all five SNPs together, patients with genotype scores ≥ 5 had a BONJ event rate of 57%; those with scores < 5 had a rate of 10%. The adjusted odds ratio was 11.2 (95% confidence interval of 1.8-69.9; p value 0.0097). Smoking, type of BP and combined genotype score of COL1A1, RANK, MMP2, OPG and OPN were significantly associated with BONJ in MM patients undergoing BP therapy.

Topics: Adult; Aged; Aryl Hydrocarbon Hydroxylases; Bone Density Conservation Agents; Cohort Studies; Collagen Type I; Collagen Type I, alpha 1 Chain; Cytochrome P-450 CYP2C8; Diphosphonates; Female; Gene Frequency; Humans; Imidazoles; Injections, Intravenous; Jaw Diseases; Male; Matrix Metalloproteinase 2; Middle Aged; Multiple Myeloma; Osteonecrosis; Osteopontin; Osteoprotegerin; Pamidronate; Polymorphism, Genetic; Polymorphism, Single Nucleotide; Receptor Activator of Nuclear Factor-kappa B; Risk Factors; Smoking; Time Factors; Tumor Necrosis Factor-alpha; Zoledronic Acid

An essential cytokine system for the osteoclast biology in multiple myeloma (MM) consists of the receptor of activator of NF-κB ligand (RANKL), its receptor (RANK), and the soluble decoy receptor, osteoprotegerin (OPG). Myeloma cells cause imbalance in OPG/RANKL interactions. We measured serum levels of OPG, soluble (s) RANKL, sRANKL/OPG ratio, markers of disease activity [LDH, CRP, interleukin-6 (IL-6), β2-microglobulin (B2M)], and angiogenic factors [hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF)], in 54 newly diagnosed MM patients and in 25 of them in plateau phase. All the above values were higher in MM patients compared to controls and decreased in plateau phase. sRANKL and RANKL/OPG were higher with advancing disease stage and skeletal grade. Significant correlations were found among RANKL and RANKL/OPG with HGF, LDH, VEGF, IL-6, and B2M. In conclusion, RANKL and OPG play significant roles in MM pathophysiology, as regulators of bone turnover and mediators of angiogenesis.

Topics: Aged; Aged, 80 and over; Cytokines; Female; Humans; Male; Middle Aged; Multiple Myeloma; Neovascularization, Pathologic; Osteoprotegerin; RANK Ligand

The study aimed at evaluating the relation of 7 parameters associated with the internal biological properties of myeloma cells and the bone marrow microenvironment to multiple myeloma (MM) stages, distinguishing its initial/asymptomatic phase from monoclonal gammopathy of undetermined significance (MGUS) and assessing their relation to myeloma prognosis. In the studied group comprising 286 individuals (89 MGUS and 179 MM patients), statistically significant differences (Mann-Whitney test) between MGUS and MM at the time of diagnosis were found in the serum levels of HGF (hepatocyte growth factor), VEGF (vascular endothelial growth factor), ICTP (intercellular - carboxy-terminal telopeptide of type I collagen), PINP (procollagen type I N-terminal propeptide), OPG (osteoprotegerin) and syndecan-1/CD138, but not in Fas. Multivariate analysis (logistic regression) revealed an unsatisfactory potential of all the 7 studied indicators to discriminate between MGUS and MM. A deeper analysis showed statistically significant differences between MGUS and the initial/asymptomatic phase of MM (stage 1 according to the International Staging System) only in the cases of syndecan-1 (p=0.001) and Fas (p=0.008). The assessment of initial values of HGF, VEGF, ICTP, PINP, OPG, syndecan-1 and Fas showed a statistically significant relation (log rank test) to the overall survival (OS) in a group of 132 patients treated with conventional chemotherapy only in the cases of syndecan-1 (p=0.0002) and Fas (p=0.018), but in none of the investigated parameters in a group of 74 patients treated with HDT/ASCT (high-dose therapy/autologous stem cell transplantation). The analysis showed that, despite significant differences in serum levels of 6 of the 7 studied parameters found between MGUS and MM, none of the markers may be included in the spectrum of indicators used to distinguish the two conditions. Despite the positive relation, especially of syndecan-1 and, to a lesser extent, of Fas to the OS in patients treated with conventional chemotherapy, these prognostic factors are not applicable to HDT/ASCT.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Collagen Type I; Diagnosis, Differential; Enzyme-Linked Immunosorbent Assay; Female; Hepatocyte Growth Factor; Humans; Male; Middle Aged; Monoclonal Gammopathy of Undetermined Significance; Multiple Myeloma; Neoplasm Staging; Osteoprotegerin; Peptide Fragments; Peptides; Procollagen; Prognosis; Syndecan-1; Vascular Endothelial Growth Factor A

The interaction of multiple myeloma (MM) cells and bone marrow stromal cells (BMSCs) induces profound changes in the bone marrow environment, influencing osteoclastogenesis and MM cell survival. Differences in receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG) production in BMSCs derived from MM patients and control subjects and the apoptosis of BMSCs and MM cells in co-cultures of both cell types were examined. RANKL and OPG expressions were examined by ELISA and semiquantitative RT-PCR. Apoptosis of BMSCs after contact with RPMI8226 and U266 cells was measured by flow cytometry and the level of ALP activity by the spectrophotometric method. OPG production by BMSCs was significantly inhibited after direct contact with RPMI8226 cells. Production of soluble RANKL was enhanced and the increase was more significant in the BMSCs of the MM patients than in those of the controls. In co-cultures of BMSCs and MM cells, significant apoptosis was detected with a concomitant decrease in ALP activity. This apoptosis decreased significantly in the presence of RANK-Fc, an antagonist of RANKL. Disturbances in the RANKL/OPG system are more profound in the BMSCs of MM patients than in those of control subjects after direct contact with RPMI8226 cells. Moreover, direct contact with RPMI8226 and U266 cells induces apoptosis of BMSCs which is mediated by an overproduction of RANKL.

Topics: Adult; Aged; Alkaline Phosphatase; Apoptosis; Bone Marrow Cells; Case-Control Studies; Cell Line, Tumor; Coculture Techniques; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Humans; Male; Middle Aged; Multiple Myeloma; Osteoprotegerin; Polymerase Chain Reaction; RANK Ligand; Recombinant Fusion Proteins; Spectrophotometry; Stromal Cells; Time Factors

This study was purposed to investigate the relationship between the levels of soluble receptor activator of nuclear factor kappa B ligand (sRANKL) and osteoprotegerin (OPG) in serum of the patients with multiple myeloma (MM) and multiple myeloma bone disease (MBD). The serum levels of sRANKL, OPG, tartrate-resistant acid phosphatase-5b (TRAP-5b) and C-terminal telopeptide of collagen I (CTP-I) which both are indexes for metabolism of osteoclast (OC) in newly diagnosed MM patients (n=42, experimental group) and healthy persons (n=25, control group) were detected by enzyme-linked immunosorbent assay. The roentgenography was used to determine bone damage in MM patients at the same time. According to these results acquired, the correlation of sRANKL/OPG ratio with levels of TRAP-5b/CTP-I, the incidence and degree of bone destruction were analyzed. The results indicated that the level of sRANKL (median value 9.33 microg/L) increased and level of OPG (median value 4.93 microg/L) decreased and the sRANKL/OPG ratio (2.65) increased significantly in experimental group. Compared with control group, the differences in all the corresponding indicators were statistically significant (p<0.05). The sRANKL/OPG ratio was closely related to levels of TRAP-5b (r=0.512, p<0.05) and CTP-I (r=0.481, p<0.05) in MM patients. After all patients in experimental groups were divided into group with bone destruction (n=29) and without bone destruction (n=13), the sRANKL/OPG ratio in the group with bone destruction was 5.13 and much higher than that in group without bone destruction (1.12) (p<0.05). A close correlation between the sRANKL/OPG ratio and degree of bone destruction (r=0.445, p<0.05) was acquired when all MM patients were divided into three groups according to degree of bone destruction, but no difference between the ratio and clinical classification and International Staging System (ISS) in MM patients was found. It is concluded that the sRANKL/OPG ratio in serum of MM patients is significantly elevated, which may be closely related to increase metabolism of OC along with the incidence and degree of bone destruction. In short, the sRANKL/OPG ratio can be used as a reference index for the diagnosis of MBD.

Topics: Adult; Aged; Bone Diseases; Case-Control Studies; Female; Humans; Male; Middle Aged; Multiple Myeloma; Osteoprotegerin; RANK Ligand

Multiple myeloma (MM) is a plasma cell malignancy characterized by a high capacity to induce osteolytic bone lesions. MM patients with osteolytic bone lesions have lower numbers of osteoblasts and decreased bone formation, which plays a critical role in the bone-destructive process. Although the mechanism of estrogen action on bone cells and myeloma cells has been widely investigated, estrogen action on bone cells in MM is unknown. In this study, the effects of the gonadal hormone 17beta-estradiol on cell growth, alkaline phosphatase (ALP) activity, mineralization capacity, and RANKL/OPG ratios in primary rat osteoblasts cultured with MM cell conditioned medium (CM) or co-cultured with RPMI8226 cells were investigated. Treatments of 10(-2) to 10 nM 17beta-estradiol reversed inhibition of proliferation and ALP activity of osteoblasts by myeloma cells in a dose-dependent manner, and 10(-2) to 1 nM 17beta-estradiol reversed inhibition of the mineralization capacity of osteoblasts by myeloma cells. In co-culture experiments with primary rat osteoblasts and myeloma cells, treatments of 10(-2) to 10 nM 17beta-estradiol down-regulated transcription and secretion of RANKL and up-regulated transcription and secretion of OPG in the osteoblasts, reversing the effects of co-cultured myeloma cells. These findings suggest that 17beta-estradiol may temper the inhibitory effects of myeloma cells on osteoblasts and improve RANKL/OPG balance, providing a new agent for treatment of bone disease in myeloma.

Topics: Alkaline Phosphatase; Animals; Base Sequence; Calcification, Physiologic; Cell Division; Coculture Techniques; Culture Media, Conditioned; DNA Primers; Estradiol; Humans; Multiple Myeloma; Osteoblasts; Osteoprotegerin; RANK Ligand; Rats; Reverse Transcriptase Polymerase Chain Reaction

To evaluate the expression of NF-kappaB pathway genes in total bone marrow samples obtained from MM at diagnosis using real-time quantitative PCR and to evaluate its possible correlation with disease clinical features and survival.. Expression of eight genes related to NF-kappaB pathway (NFKB1, IKB, RANK, RANKL, OPG, IL6, VCAM1 and ICAM1) were studied in 53 bone marrow samples from newly diagnosed MM patients and in seven normal controls, using the Taqman system. Genes were considered overexpressed when tumor expression level was at least four times higher than that observed in normal samples.. The percentages of overexpression of the eight genes were: NFKB1 0%, IKB 22.6%, RANK 15.1%, RANKL 31.3%, OPG 7.5%, IL6 39.6%, VCAM1 10% and ICAM1 26%. We found association between IL6 expression level and International Staging System (ISS) (p=0.01), meaning that MM patients with high ISS scores have more chance of overexpression of IL6. The mean value of ICAM1 relative expression was also associated with the ISS score (p=0.02). Regarding OS, cases with IL6 overexpression present worse evolution than cases with IL6 normal expression (p=0.04).. We demonstrated that total bone marrow aspirates can be used as a source of material for gene expression studies in MM. In this context, we confirmed that IL6 overexpression was significantly associated with worse survival and we described that it is associated with high ISS scores. Also, ICAM1 was overexpressed in 26% of cases and its level was associated with ISS scores.

Topics: Adult; Aged; Aged, 80 and over; Bone Marrow Cells; Case-Control Studies; DNA-Binding Proteins; Female; Gene Expression; Humans; I-kappa B Proteins; Intercellular Adhesion Molecule-1; Interleukin-6; Male; Middle Aged; Multiple Myeloma; NF-kappa B; NF-kappa B p50 Subunit; NF-KappaB Inhibitor alpha; Osteoprotegerin; Pilot Projects; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Vascular Cell Adhesion Molecule-1

Receptor activator of nuclear factor-kappaB ligand (RANKL) plays a key role in osteoclast activation in myeloma bone disease. The increased expression of RANKL in the bone marrow microenvironment was demonstrated in several studies, but there are only rare data on circulating RANKL levels in patients with multiple myeloma (MM).. In the current study, we investigated the clinical significance of serum RANKL levels, using an enzyme-linked immunosorbent assay test that detects both free and osteoprotegerin (OPG)-bound RANKL (total-RANKL, tRANKL) in patients with newly diagnosed MM (n = 93) and monoclonal gammopathy of undetermined significance (MGUS; n = 20) compared with healthy controls (n = 20).. Circulating serum tRANKL was significantly elevated in patients with MM compared with controls (P < .001) or MGUS (P < .001). Furthermore, tRANKL levels were higher in smoldering MM versus MGUS (P = .031) and in symptomatic versus smoldering MM (P < .001). Serum tRANKL increased parallel to International Staging System stages I to III (P = .004) and correlated with the presence of lytic bone lesions (P < .001). Total-RANKL was a prognostic factor for overall survival in symptomatic MM (P = .043). A significantly longer progression-free survival was observed in patients with a > 50% decrease in tRANKL levels after 3 months of combined chemotherapy and bisphosphonate treatment.. Our study demonstrates for the first time that serum tRANKL reflects advanced disease, lytic bone destruction, and poor prognosis in MM.

Topics: Adult; Aged; Aged, 80 and over; Enzyme-Linked Immunosorbent Assay; Female; Humans; Male; Middle Aged; Multiple Myeloma; Osteoprotegerin; Prognosis; RANK Ligand

Bone biology and the molecular basis of normal bone remodeling are important for understanding bone health, particularly for medical professionals who are treating patients with skeletal complications. Key factors in bone remodeling include RANK ligand, which stimulates bone resorption, and osteoprotegerin (OPG), which inhibits bone resorption. The ratio between these two factors regulates osteoclast formation and activity. An imbalance in the expression of RANK ligand and OPG is an underlying mechanism in cancer treatment-induced bone loss, in bone metastasis in patients with solid tumors, and in osteolysis in patients with multiple myeloma. In cancer-induced bone disease, for example, RANK ligand overwhelms the effects of OPG, leading to imbalanced bone remodeling and the "vicious cycle" of metastatic disease. Experimental therapeutics that target RANK ligand are increasingly being studied and have shown promise for decreasing tumor-related bone disease.

Topics: Animals; Antineoplastic Agents; Bone Diseases, Metabolic; Bone Neoplasms; Bone Remodeling; Humans; Mice; Multiple Myeloma; Osteoprotegerin; RANK Ligand

Osteolytic bone disease (OBD) in multiple myeloma (MM) is caused by interactions between MM cells and the bone marrow microenvironment and is characterized by increased osteoclastic bone resorption and decreased osteoblastic bone formation. Recently, the role of osteoblast inhibition has come into focus, especially the possible role of overexpression of DKK1, an inhibitor of the Wnt signalling pathway. Further, CKS2, PSME2 and DHFR have also been reported as candidate genes for OBD. We studied the gene expression by quantitative reverse transcription polymerase chain reaction of TNFSF11 (RANKL), TNFSF11A (RANK), TNFRSF11B (OPG), CCL3 (MIP1A), CCL4 (MIP1B), PTHR1 (PTHrp), DKK1, CKS2, PSME2 and DHFR in purified, immunophenotypic FACS-sorted plasma cells from 171 newly diagnosed MM patients, 20 patients with monoclonal gammopathy of undetermined significance and 12 controls. The gene expressions of the analysed genes were correlated with radiographically assessed OBD. Only overexpression of DKK1 was correlated to the degree of OBD. Myeloma cells did not express TNFSF11A, TNFSF11, or TNFRSF11B, and very rarely expressed CCL3 and PTHR11. CCL4, CKS2, PSME2 and DHFR were variably expressed, but the expression of these genes showed no correlation with OBD. In contrast, loss of PSME2 expression in MM plasma cells was significantly correlated with OBD.

Topics: Adult; Aged; Aged, 80 and over; Carrier Proteins; CDC2-CDC28 Kinases; Cell Cycle Proteins; Chemokine CCL3; Chemokine CCL4; Female; Gene Expression; Humans; Intercellular Signaling Peptides and Proteins; Male; Middle Aged; Multiple Myeloma; Muscle Proteins; Osteolysis; Osteoprotegerin; Plasma Cells; Proteasome Endopeptidase Complex; Protein Kinases; Receptor Activator of Nuclear Factor-kappa B; Receptor, Parathyroid Hormone, Type 1; Tetrahydrofolate Dehydrogenase

Multiple myeloma (MM) is characterized by osteolytic bone lesions (OBL) that arise as a consequence of osteoblast inactivation and osteoclast activation adjacent to tumor foci within bone. Wnt signaling in osteoblasts regulates osteoclastogenesis through the differential activation and inactivation of Receptor Activator of Nuclear factor Kappa B Ligand (RANKL) and osteoprotegerin (OPG), positive and negative regulators of osteoclast differentiation, respectively. We demonstrate here that MM cell-derived DKK1, a soluble inhibitor of canonical Wnt signaling, disrupted Wnt3a-regulated OPG and RANKL expression in osteoblasts. Confirmed in multiple independent assays, we show that pretreatment with rDKK1 completely abolished Wnt3a-induced OPG mRNA and protein production by mouse and human osteoblasts. In addition, we show that Wnt3a-induced OPG expression was diminished in osteoblasts cocultured with a DKK1-expressing MM cell line or primary MM cells. Finally, we show that bone marrow sera from 21 MM patients significantly suppressed Wnt3a-induced OPG expression and enhanced RANKL expression in osteoblasts in a DKK1-dependent manner. These results suggest that DKK1 may play a key role in the development of MM-associated OBL by directly interrupting Wnt-regulated differentiation of osteoblasts and indirectly increasing osteoclastogenesis via a DKK1-mediated increase in RANKL-to-OPG ratios.

Topics: Animals; Base Sequence; Cell Differentiation; Cell Line; Cell Line, Tumor; Coculture Techniques; DNA Primers; Gene Expression; Gene Silencing; Humans; Intercellular Signaling Peptides and Proteins; Mice; Multiple Myeloma; Osteoblasts; Osteolysis; Osteoprotegerin; RANK Ligand; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Transfection; Tumor Cells, Cultured; Wnt Proteins; Wnt3 Protein; Wnt3A Protein

The growth and survival of myeloma cells is critically regulated by cells of the bone marrow microenvironment, including osteoblasts. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of myeloma cell apoptosis, however, this antimyeloma activity is inhibited by osteoprotegerin (OPG) released from osteoblasts. Therefore, we hypothesized that specific agonists of TRAIL death receptors would not be inhibited by OPG released from osteoblasts and thus may represent a novel therapeutic approach in multiple myeloma. In the present study, TRAIL-induced apoptosis was demonstrated to be mediated through both DR4 and DR5. Specific agonist antibodies to DR4 or DR5 dose-dependently induced myeloma cell apoptosis, which was not prevented by OPG or by medium conditioned by osteoblasts. Co-culture of myeloma cells with osteoblasts protected against TRAIL-induced apoptosis of myeloma cells, and this protective effect was due to OPG. In contrast, the co-culture of myeloma cells with osteoblasts had no protective effect on apoptosis induced by specific agonists of DR4 or DR5. TRAIL has been proposed as a potential antitumour therapy, but within the bone marrow microenvironment OPG may interfere with the action of TRAIL. Specific agonists of TRAIL death receptors would not be subject to this inhibition and thus may provide an alternative specific antimyeloma therapy.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Bone Marrow Cells; Cell Line, Tumor; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Multiple Myeloma; Osteoblasts; Osteoprotegerin; Receptors, TNF-Related Apoptosis-Inducing Ligand; Reverse Transcriptase Polymerase Chain Reaction

Serum levels of OPG and RANKL and their clinical correlations were analyzed in 66 newly-diagnosed patients with multiple myeloma (MM). RANKL and RANKL /OPG ratio were significantly increased in advanced clinical stages and high grade myeloma bone disease (MBD), while OPG showed a tendency to decrease. Renal failure modified the expression of OPG. RANKL and RANKL/OPG ratios are informative markers for myeloma tumor burden and MBD.

Topics: Biomarkers; Bone Neoplasms; Humans; Multiple Myeloma; Osteoprotegerin; RANK Ligand; Renal Insufficiency; Tumor Burden

We describe a new model of myeloma bone disease in which beta2m NOD/SCID mice injected with KMS-12-BM cells develop medullary disease after tail vein administration. Micro-computed tomography analysis demonstrated significant bone loss in the tibiae and vertebrae of diseased animals compared to controls, with loss of cortical bone (P<0.01), as well as trabecular bone volume, thickness and number (P<0.05 for all). Bone marrow of diseased animals demonstrated an increase in osteoclasts (P<0.01) and reduction in osteoblasts (P<0.01) compared to control animals. Both bone loss and osteoclast increase correlated with the degree of disease involvement. Mesenchymal stem cells (MSCs) were lentivirally transduced to express human osteoprotegerin (hOPG). Systemic administration of OPG expressing MSC reduced osteoclast activation (P<0.01) and trabecular bone loss in the vertebrae (P<0.05) and tibiae of diseased animals, to levels comparable to non-diseased controls. Because of its predominantly medullary involvement and quantifiable parameters of bone disease, the KMS-12-BM xenogeneic model provides unique opportunities to test therapies targeted at the bone marrow microenvironment.

Topics: Animals; Bone and Bones; Cell Line; Disease Models, Animal; Genetic Therapy; Humans; Kinetics; Lentivirus; Mesenchymal Stem Cells; Mice; Mice, Inbred NOD; Mice, SCID; Multiple Myeloma; Neoplasm Transplantation; Osteoblasts; Osteoclasts; Osteoprotegerin; Tibia

Osteoprotegerin (OPG) binds the ligand for receptor activator of nuclear factor kappaB (RANKL) to prevent association with its receptor RANK and inhibit osteoclast-mediated bone resorption. OPG has been reported, recently, to inhibit tumor necrosis factor-related apoptosis-induced ligand (TRAIL)-induced tumor cell apoptosis. This raises the possibility that OPG may play a unique role in regulating these two signaling pathways. However, there are little data on the interactions between OPG, RANKL, and TRAIL, and the relative affinity of OPG for these two ligands is unknown. In the present study we examined the ability of OPG to bind native human TRAIL and RANKL under physiological conditions. Native TRAIL was expressed in Escherichia coli, purified to homogeneity, and shown to induce human myeloma cell apoptosis. OPG inhibited native TRAIL from binding the TRAILR1 at 37 degrees C in vitro. Similarly, OPG prevented RANKL from binding to RANK. TRAIL also prevented OPG-mediated inhibition of RANKL from binding RANK. The affinity of OPG for native TRAIL and RANKL at 37 degrees C was determined by plasmon surface resonance analysis. OPG had a binding affinity for TRAIL of 45 nM, whereas the affinity of OPG for RANKL was 23 nM. These data suggest that OPG can bind both RANKL and TRAIL and that the affinity of OPG for these two ligands is of a similar order of magnitude. Furthermore, OPG prevented TRAIL-mediated reductions in cell viability, whereas TRAIL inhibited OPG-mediated inhibition of osteoclastogenesis in vitro. This highlights the pivotal role of OPG in regulating the biology of both RANKL and TRAIL.

Topics: Apoptosis; Cell Line, Tumor; Cloning, Molecular; Escherichia coli; Humans; Models, Biological; Multiple Myeloma; Osteoprotegerin; Protein Binding; Protein Structure, Tertiary; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Recombinant Proteins; Sequence Analysis, DNA; Solubility; Surface Plasmon Resonance; TNF-Related Apoptosis-Inducing Ligand

To investigate the effects of myeloma cells on the differentiation of osteoclast precursors (pOCs) into OCs in different culture systems in vitro and the interaction between OCs and myeloma cells.. Myeloma cell lines 8226, XG1 and XG7 and pOCs were cocultured in different culture system. OCs was examined by TRAP staining. RT-PCR was used to evaluate the expression of receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG) of myeloma cells and the effects of myeloma cells on RANKL/OPG expression in coculture. The role of OCs in myeloma cells cycle was measured by FCM with PI staining. The supportive effects of OCs on myeloma cells survival were determined by FCM with double staining for annexin V and PI.. 8226 and XG1 cells could directly stimulate the differentiation of pOCs into TRAP+ multinuclear mature OCs. Myeloma cells, which expressed neither RANKL nor OPG, upregulated RANKL expression and decreased OPG expression in mouse primary bone marrow stromal cells (pBMSC). When OCs were co-cultured with myeloma cells, all OCs apparently remained alive after 7 days while devoid of sRANKL and M-CSF. OCs stimulated the proliferation of myeloma cells in co-culture systems,the cell number increased to (3.8 +/- 0.1) x 10(5)/well, (3.9 +/- 0.1) x 10(5)/well, (4.0 +/- 0.1) x 10(5)/well, and to (8.7 +/- 0.1) x 10(5)/well, (9.1 +/- 0.1) x 10(5)/well, (9.0 +/- 0.1 ) x 10(5)/well after co-culture for 3 days and 7 days for XG1 cells, XG7 cells and 8226 cells, respectively (P <0.01). However, OCs could counteract cytotoxic effects of dexamethasone. The proportion of Annexin V-/PI- cells were 57.71%, 82.18% and 90.92% for 8226 cells, XG1 and XG7 cells after co-culture with OCs (P <0.01).. Myeloma cells stimulated the differentiation of pOCs into TRAP+ multinuclear mature OCs by directly and/or indirectly disrupting the balance of RANKL/OPG, OCs promoted MM cells growth and survival, thus maintaining a vicious circle between myeloma cells and osteoclasts.

Topics: Animals; Bone Marrow Cells; Cell Communication; Cell Line, Tumor; Cell Proliferation; Cell Survival; Coculture Techniques; Humans; Mice; Multiple Myeloma; Osteoclasts; Osteoprotegerin; RANK Ligand; Stromal Cells

Multiple myeloma (MM) is characterized by accumulation of monoclonal plasma cells in the bone marrow and progression of lytic bone lesions. MM cells enhance bone resorption by triggering a coordinated increase in RANK ligand and decrease in osteoprotegerin in the bone marrow. Macrophage inflammatory protein (MIP)-1alpha and (MIP)-1beta are secreted by MM cells, and play a major role in the enhancement of bone resorption by MM cells. Furthermore, the growth and survival of MM cells are enhanced by contact with osteoclasts (OCs) suggesting the presence of a vicious cycle between OCs and MM cells. OCs also enhance angiogenesis in concert with MM cells largely through the cooperative actions of osteopontin from OCs and VEGF from MM cells. The angiogenic effect may further facilitate the vicious cycle between bone destruction and MM cell expansion. In addition, MM cells secrete soluble factor(s) to suppress bone formation. Secreted Frizzled-related protein (sFRP)-2, an inhibitor of Wingless type (Wnt) binding to Frizzled, is produced by most MM cells, and immunodepletion of sFRP-2 abrogates the inhibition of bone formation. Thus, MM cells enhance bone resorption and suppress bone formation to cause destructive bone lesions. Further elucidation of the mechanism of bone destruction by MM may lead to a novel therapeutic approach to prevent bone destruction and tumor growth.

Topics: Bone Development; Bone Marrow Cells; Bone Resorption; Carrier Proteins; Cell Division; Cell Survival; Glycoproteins; Humans; Membrane Glycoproteins; Multiple Myeloma; NF-kappa B; Osteoclasts; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Multiple myeloma (MM) is often associated with an increased osteoclast (OC) activity. Using an in vitro osteoclastogenesis model consisting of MM unstimulated and unfractionated peripheral blood mononuclear cells (PBMCs), we showed that T cells support OC formation and survival. Differently, in T cell-depleted MM PBMC cultures, exogenous macrophage-colony stimulating factor (M-CSF) and receptor activated of nuclear factor-kappaB ligand (RANKL) were necessary for osteoclastogenesis. We found RANKL, OPG, and TRAIL overexpression by fresh MM T cells. Despite high osteoprotegerin (OPG) levels, the persistence of osteoclastogenesis can be related to the formation of the OPG/TRAIL complex. Our results highlight that MM T cells support OC formation and survival, possibly involving OPG/TRAIL interaction.

Topics: Apoptosis Regulatory Proteins; Bone Diseases; Cells, Cultured; Female; Glycoproteins; Humans; Male; Membrane Glycoproteins; Models, Biological; Multiple Myeloma; Osteoclasts; Osteoprotegerin; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

The effect of bortezomib on bone remodelling was evaluated in 34 relapsed myeloma patients. At baseline, patients had increased serum concentrations of dickkopf-1 (DKK-1), soluble receptor activator of nuclear factor-kappaB ligand (sRANKL), sRANKL/osteoprotegerin ratio, C-telopeptide of type-I collagen (CTX) and tartrate-resistant acid phosphatase isoform-5b (TRACP-5b); bone-alkaline phosphatase and osteocalcin were reduced. Serum DKK-1 correlated with CTX and severe bone disease. Bortezomib administration significantly reduced serum DKK-1, sRANKL, CTX, and TRACP-5b after four cycles, and dramatically increased bone-alkaline phosphatase and osteocalcin, irrespective of treatment response. This is the first study showing that bortezomib reduces DKK-1 and RANKL serum levels, leading to the normalisation of bone remodelling in relapsed myeloma.

Topics: Acid Phosphatase; Adult; Aged; Aged, 80 and over; Alkaline Phosphatase; Biomarkers; Bone Remodeling; Boronic Acids; Bortezomib; Case-Control Studies; Collagen Type I; Diphosphonates; Female; Humans; Imidazoles; Intercellular Signaling Peptides and Proteins; Isoenzymes; Male; Middle Aged; Multiple Myeloma; Osteocalcin; Osteoprotegerin; Peptides; Protease Inhibitors; Pyrazines; RANK Ligand; Recurrence; Statistics, Nonparametric; Tartrate-Resistant Acid Phosphatase; Zoledronic Acid

We evaluated the effect of zoledronic acid (ZA) on serum levels of osteoprotegerin (OPG) and the ligand for receptor activator of nuclear factor kappaB (RANKL) in patients with smoldering myeloma. In treated subjects we found an increase of OPG accounting for an effect of ZA on osteoblast and/or bone marrow stromal cells together with the direct effect on osteoclasts.

Topics: Diphosphonates; Humans; Imidazoles; Multiple Myeloma; Osteoprotegerin; Zoledronic Acid

Topics: Animals; Carrier Proteins; Disease Models, Animal; Glycoproteins; Humans; Membrane Glycoproteins; Multiple Myeloma; Osteoblasts; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Immunoglobulin (Ig) M myeloma is a distinct entity with features of multiple myeloma (MM) and Waldenstrom's macroglobulinemia (WM). The malignant cells in IgM myeloma have a distinctive chromosomal translocation that differentiates them from WM. These cells are postgerminal-center in origin with isotype-switch transcripts. They appear to be arrested at a point of maturation between that of WM and MM. Preliminary data indicate that a pattern of osteoclast-activating factor and osteoprotegerin expression similar to that observed in classic MM is present in IgM myeloma. Additional studies on patients with this rare tumor may provide further insight into the pathogenesis of bone disease in plasma cell dyscrasias.

Topics: Cell Differentiation; Cytokines; Glycoproteins; Humans; Immunoglobulin M; Lymphokines; Male; Middle Aged; Multiple Myeloma; Osteoprotegerin; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Translocation, Genetic; Waldenstrom Macroglobulinemia

Topics: Biomarkers; Glycoproteins; Humans; Multiple Myeloma; Osteoprotegerin; Prognosis; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Survival Rate

Multiple myeloma is a plasma cell malignancy characterized by the development of osteolytic lesions leading to bone pain, pathologic fractures, and hypercalcemia. Osteoprotegerin (OPG) is a potent inhibitor of osteoclast differentiation and activation, but is limited as a therapeutic agent due to its short circulating half-life. In order to overcome these limitations, the therapeutic effects of native OPG gene transfer are examined.. We used replication-incompetent lentiviral vectors to transfer the unmodified, native human OPG gene ex vivo into human ARH-77 cells injected into severe combined immunodeficient (SCID) mice, to determine gene transfer efficiency as well as the impact on disease progression in this in vivo model.. We can efficiently transfer and express either the LacZ marker gene or the native human OPG gene into human ARH-77 cells. Moreover, transfer of the OPG gene into ARH-77 cells reduces the development of osteolytic bony lesions when these cells are injected into SCID mice, compared to mice injected with either unmodified ARH-77 cells or ARH-77 cells transduced with the OPG gene in the antisense orientation. This therapeutic effect was manifested as a reduction in vertebral compression deformities and in the number and size of long-bone osteolytic lesions on skeletal radiographs, as well as a decrease in osteoclast surface on histologic analysis.. A lentiviral vector can efficiently transfer the native human OPG gene to myeloma cells ex vivo and inhibit myeloma-induced bone destruction, thereby suggesting a therapeutic potential for unmodified, native OPG gene transfer for osteoclast-dependent skeletal disorders.

Topics: Animals; Bone Density; Cell Line, Tumor; DNA, Antisense; DNA, Recombinant; Genetic Therapy; Genetic Vectors; Glycoproteins; Lac Operon; Lentivirus; Mice; Multiple Myeloma; Neoplasm Transplantation; Osteoclasts; Osteolysis; Osteoprotegerin; Pilot Projects; Radiography; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Transduction, Genetic; Xenograft Model Antitumor Assays

Neovascularisation and bone resorption are related to myeloma disease activity.. To investigate the possible prognostic importance of serum syndecan-1, basic fibroblast growth factor (bFGF) and osteoprotegerin (OPG) levels, the relationship between them, with parameters of disease activity and the effect of treatment on their levels.. Twenty-seven patients were studied from diagnosis and an additional five from remission, for a median follow-up of 40 months. Twenty-three patients received chemotherapy plus bisphosphonates and nine only bisphosphonates. Sera from 11 healthy individuals (HI) were used as controls. Cytokines were determined by commercially available enzyme-linked immunosorbent assays (ELISA) kits.. In HI, median syndecan-1 was 40 ng/mL (28-75), bFGF 8 pg/mL (7-30), OPG 35 pg/mL (4-100). Pretreatment median serum syndecan-1 was 177.5 ng/mL (34-3500), bFGF 11.5 pg/mL (8-65) and OPG 100 pg/mL (4-1000). Pretreatment syndecan-1, bFGF and OPG serum levels were increased in patients compared with HI (P = 0.001, 0.03 and 0.01, respectively). Syndecan-1 and bFGF levels were correlated with stage (P = 0.004 and 0.03, respectively). Both syndecan-1 and OPG levels were correlated with beta2M (P = 0.04 and 0.01, respectively). Patients with elevated syndecan-1 and bFGF serum levels had shorter survival than patients with normal levels (P = 0.01 and 0.05, respectively). After chemotherapy syndecan-1 and OPG levels were found to be decreased in responders and syndecan-1 level was reduced in patients receiving bisphosphonates alone.. Pretreatment syndecan-1, bFGF and OPG levels were found to be increased at diagnosis. Syndecan-1 and OPG fluctuated according to MM activity. Elevated serum syndecan-1 and bFGF levels predicted short survival.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; beta 2-Microglobulin; Biomarkers, Tumor; Bone Resorption; Cyclophosphamide; Dexamethasone; Diphosphonates; Doxorubicin; Female; Fibroblast Growth Factor 2; Follow-Up Studies; Glycoproteins; Humans; Life Tables; Male; Melphalan; Membrane Glycoproteins; Middle Aged; Multiple Myeloma; Neoplasm Proteins; Neoplasm Staging; Neovascularization, Pathologic; Osteoprotegerin; Pamidronate; Prednisone; Prognosis; Proteoglycans; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Survival Analysis; Syndecan-1; Syndecans; Vincristine

The osteoprotegerin (OPG)/receptor activator of NF-kappa B ligand (RANKL) system has a major role in the pathogenesis of bone disease in myeloma (MM). The effect of autologous stem cell transplantation (ASCT) on bone turnover in MM was evaluated in 51 patients (35M/16F). Markers of bone resorption (NTX, TRACP-5b), bone formation (bone-alkaline phosphatase (bALP), osteocalcin), OPG and sRANKL were measured pre- and every month post-ASCT. The median follow-up period was 12 months. Four patients were transplanted in CR, 44 were transplanted in PR and three patients had progressive/resistant disease. All patients received bisphosphonates both pre- and post-ASCT. At baseline the majority of patients had increased NTX, TRACP-5b levels, and sRANKL/OPG ratio, while markers of bone formation were strongly suppressed. ASCT produced a significant reduction of sRANKL/OPG ratio, with a concomitant decrease of NTX, and TRACP-5b levels, starting the second month post-ASCT. Bone formation markers, osteocalcin and bALP, started to increase after the 9th and 11th month post-ASCT, respectively, while the increase of OPG preceded this. These results provide biochemical evidence that ASCT normalizes the abnormal bone resorption in MM patients possibly through the decrease of RANKL/OPG ratio, while bone formation requires a longer period to return to normal.

Topics: Adult; Aged; Biomarkers; Bone Diseases; Bone Remodeling; Bone Resorption; Carrier Proteins; Case-Control Studies; Diphosphonates; Female; Follow-Up Studies; Glycoproteins; Hematopoietic Stem Cell Transplantation; Humans; Male; Membrane Glycoproteins; Middle Aged; Multiple Myeloma; Osteogenesis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Time Factors; Transplantation, Autologous

Topics: Adult; Age Distribution; Aged; Aged, 80 and over; Case-Control Studies; Enzyme-Linked Immunosorbent Assay; Female; Glycoproteins; Humans; Hypergammaglobulinemia; Male; Middle Aged; Multiple Myeloma; Osteoprotegerin; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Sex Distribution

The development of multiple myeloma (MM) bone disease is mediated by increased number and activity of osteoclasts (OCs). Using an in vitro osteoclastogenesis model consisting of unstimulated and unfractionated peripheral blood mononuclear cells (PBMCs) from patients with MM, we showed that T cells support the formation of OCs with longer survival. Different from T-cell-depleted MM PBMC cultures, exogenous macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor-kappaB ligand (RANKL) were necessary for the formation of OCs; however, they did not exhibit longer survival. We found up-regulated production of RANKL, osteoprotegerin (OPG), and TNF-related apoptosis-inducing ligand (TRAIL) by fresh MM T cells. Despite high OPG levels, the persistence of osteoclastogenesis can be related to the formation of the OPG/TRAIL complex demonstrated by immunoprecipitation experiments and the addition of anti-TRAIL antibody which decreases OC formation. OCs overexpressed TRAIL decoy receptor DcR2 in the presence of MM T cells and death receptor DR4 in T-cell-depleted cultures. In addition, increased Bcl-2/Bax (B-cell lymphoma-2/Bcl2-associated protein X) ratio, following Bcl-2 up-regulation, was detected in OCs generated in the presence of T cells. Our results highlight that MM T cells support OC formation and survival, possibly involving OPG/TRAIL interaction and unbalanced OC expression of TRAIL death and decoy receptors.

Topics: Aged; Aged, 80 and over; Apoptosis Regulatory Proteins; Bone Diseases; Case-Control Studies; Cell Survival; Cells, Cultured; Cytokines; Female; Glycoproteins; Humans; Male; Membrane Glycoproteins; Middle Aged; Multiple Myeloma; Osteoclasts; Osteoprotegerin; Protein Binding; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; T-Lymphocytes; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

The aim of the present study was to determine whether modifying the local bone environment with osteoprotegerin (OPG), the soluble decoy receptor for receptor activator of nuclear factor-kappaB (RANK) ligand, could affect tumor burden and survival in the 5T33MM murine model of multiple myeloma. Treatment of mice, injected with 5T33MM cells, with recombinant OPG (Fc-OPG) caused a significant decrease in serum paraprotein and tumor burden and a significant increase in time to morbidity. This was associated with a decrease in osteoclast number in vivo but had no effect on apoptosis and proliferation of 5T33MM cells in vitro. These data indicate that targeting the bone microenvironment by inhibiting the interaction between RANK ligand and RANK with Fc-OPG not only inhibits the development of myeloma bone disease but also decreases tumor growth and increases survival.

Topics: Animals; Cell Division; Disease Models, Animal; Glycoproteins; Mice; Mice, Inbred C57BL; Multiple Myeloma; Osteoclasts; Osteoprotegerin; Paraproteinemias; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Recombinant Proteins

The bone destruction in myeloma patients is largely responsible for the clinical features of the disease. However, only recently has attention focused on identifying and developing drugs targeted specifically at the osteolysis. Receptor activator of NF-kappaB ligand (RANKL), macrophage inflammatory protein (MIP)-1alpha, and proteasomal function have been implicated in the pathogenesis of myeloma and associated bone disease. We provide "proof of principle" in preclinical myeloma models that these are indeed valid molecular targets in development of novel therapeutics.. The efficacy of antagonists of RANKL and MIP-1alpha bioactivities (RANK.Fc and neutralizing monoclonal anti-MIP-1alpha antibody) in ameliorating osteolysis and reducing tumor burden was evaluated in a mouse model in which murine myeloma 5TGM1 cells are injected intravenously into syngeneic mice. In addition, the activity of a petidyl aldehyde proteasome inhibitor (proteasome inhibitor-1 [PSI]) on tumor growth was tested in a murine 5TGM1 plasmacytoma model and in mice intravenously inoculated with 5TGM1 cells.. RANK.Fc and anti-MIP-1alpha antibody inhibited the development and progression of osteolytic lesions and significantly reduced tumor load assessed by serum monoclonal paraprotein titers. Intratumoral injections of PSI inhibited growth of 5TGM1 plasmacytomas and induced tumor regression in some cases. In addition, systemic administration of PSI significantly prolonged time to onset of paraplegia in tumor-bearing mice.. The results highlight the critical roles of RANKL and MIP-1alpha in the development and progression of myeloma and provide a basis for future evaluation in myeloma patients of novel therapeutics that disrupt interactions of RANKL and MIP-1alpha with their cognate receptors. The data also suggest that further studies in preclincal myeloma models aimed at identifying other proteasome inhibitors with antitumor efficacy would be worthwhile.

Topics: Animals; Antibodies, Monoclonal; Boronic Acids; Bortezomib; Carrier Proteins; Chemokine CCL3; Chemokine CCL4; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Disease Models, Animal; Glycoproteins; Macrophage Inflammatory Proteins; Membrane Glycoproteins; Mice; Multienzyme Complexes; Multiple Myeloma; Osteolysis; Osteoprotegerin; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteins; Pyrazines; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

A major clinical feature in multiple myeloma is the development of osteolytic bone disease. The increase in bone destruction is due to uncontrolled osteoclastic bone resorption. Until recently the factors responsible for mediating the increase in osteoclast formation in myeloma have been unclear. However, recent studies have implicated a number of factors, including the ligand for receptor activator of NFkappaB (RANKL) and macrophage inflammatory protein-1alpha. The demonstration that increased osteoclastic activity plays a central role in this process and the identification of molecules that may play a critical role in the development of myeloma bone disease have resulted in studies aimed at identifying new approaches to treating this aspect of myeloma.. Studies have been performed to determine the ability of recombinant osteoprotegerin (Fc.OPG), a soluble decoy receptor for RANKL, and potent new bisphosphonates to inhibit the development of myeloma bone disease in the 5T2MM murine model of multiple myeloma.. Fc.OPG was shown to prevent the development of osteolytic bone lesions in 5T2MM bearing animals. These changes were associated with a preservation of the cancellous bone loss induced by myeloma cells and an inhibition of osteoclast formation. Bisphosphonates, including ibandronate and zoledronic acid, were also shown to inhibit the development of osteolytic bone lesions in the 5T2MM model and alternative models of myeloma bone disease.. Bisphosphonates and Fc.OPG are effective inhibitors of the development of osteolytic bone lesions in pre-clinical murine models of myeloma bone disease.

Topics: Animals; Diphosphonates; Disease Models, Animal; Glycoproteins; Mice; Mice, SCID; Multiple Myeloma; Osteoclasts; Osteolysis; Osteoprotegerin; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Myeloma cells grow only in the bone marrow closely associated with bone,suggesting that this microenvironment provides critical signals for their growth and survival. Osteoprotegerin (OPG) is a member of the tumor necrosis factor (TNF) receptor family, which binds to the ligand for receptor activator of nuclear factor kappa B and inhibits bone resorption. However, it is unclear whether OPG can also bind to other TNF family members, such as TNF-related apoptosis-inducing ligand/Apo2 ligand (TRAIL/Apo2L), and, by inhibiting their activity, function as a survival factor for myeloma cells. In the present study MG63 osteoblast-like cells and primary bone marrow stromal cells were both shown to produce OPG, whereas human myeloma cells did not produce OPG but down-regulated release of OPG from MG63 cells. TRAIL/Apo2L induced apoptosis in myeloma cells, and this could be prevented with the addition of recombinant OPG. Medium conditioned by MG63 cells was also shown to inhibit TRAIL/Apo2L-induced apoptosis, an effect that was reversed by the addition of soluble receptor activator of nuclear factor kappa B ligand. Medium conditioned by cocultures of MG63 cells with myeloma cells had a reduced effect on TRAIL/Apo2L-induced apoptosis, reflecting the decreased concentrations of OPG in cocultures of myeloma cells with bone cells. These observations suggest that OPG may function as a paracrine survival factor in the bone marrow microenvironment in multiple myeloma.

Topics: Antibodies; Apoptosis; Apoptosis Regulatory Proteins; Bone Marrow Cells; Carrier Proteins; Cell Survival; Coculture Techniques; Culture Media, Conditioned; Glycoproteins; Humans; Membrane Glycoproteins; Multiple Myeloma; Osteoblasts; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Interaction between receptor activator of nuclear factor kappaB ligand (RANKL) and RANK/osteoprotegerin (OPG) plays a dominant role in osteoclast activation and possibly in plasma cell survival in multiple myeloma (MM). We measured soluble RANKL (sRANKL), OPG, and bone remodeling markers in 121 patients with newly diagnosed MM to evaluate their role in bone disease and survival. Serum levels of sRANKL were elevated in patients with MM and correlated with bone disease. The sRANKL/OPG ratio was also increased and correlated with markers of bone resorption, osteolytic lesions, and markers of disease activity. The sRANKL/OPG ratio, C-reactive protein (CRP), and beta2-microglobulin were the only independent prognostic factors predicting survival in multivariate analysis. We generated a prognostic index based on these factors that divided our patients into 3 risk groups. The low-risk group had a 96% probability of survival at 5 years, whereas the intermediate-risk and the high-risk groups had probabilities of survival of 52% and 0%, respectively. Not only do these results confirm for the first time in humans the importance of sRANKL/OPG in the development of bone disease, they also highlight the role of this pathway in the biology of plasma cell growth as reflected by its influence on survival.

Topics: Adult; Aged; Aged, 80 and over; beta 2-Microglobulin; Biomarkers; Bone Diseases; Bone Remodeling; C-Reactive Protein; Carrier Proteins; Cell Survival; Female; Glycoproteins; Humans; Male; Membrane Glycoproteins; Middle Aged; Multiple Myeloma; Multivariate Analysis; Osteoprotegerin; Prognosis; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Severity of Illness Index; Solubility; Survival Analysis

Tartrate-resistant acid phosphatase isoform-5b (TRACP-5b), a new marker reflecting osteoclast activity, and osteoprotegerin (OPG) were measured in 121 patients with multiple myeloma (MM) at diagnosis, and in 63 of them during pamidronate administration, to define their correlation with the extent of bone disease and disease activity in MM. Radiographic evaluation of the skeleton, measurement of other markers of bone remodelling, including N-terminal cross-linking telopeptide of type-I collagen (NTX), bone alkaline phosphatase and osteocalcin and of markers of disease activity (beta2-microglobulin, paraprotein, interleukin-6 (IL-6), were also performed. Levels of TRACP-5b were increased (p <.0001), while OPG was decreased in MM patients compared to controls (p <.01). TRACP-5b levels were associated with the radiographically assessed severity of bone disease (p <.0001) as well as with levels of NTX, IL-6 and beta2-microglobulin (p <.001, for each biochemical parameter, respectively). The combination of pamidronate with VAD-chemotherapy produced a reduction in TRACP-5b, NTX, IL-6, paraprotein and beta2-microglobulin levels from the 2nd month of treatment, with no effect on bone formation and OPG. A strong correlation was observed between changes in TRACP-5b and changes in NTX, IL-6 and beta2-microglobulin, while TRACP-5b predicted the disease progression in 5 patients. These findings suggest that TRACP-5b is increased in MM, reflects the extent of myeloma bone disease and may have a predictive value. TRACP-5b has also proved to be very useful for monitoring antimyeloma treatment, which had no effect on OPG levels.

Topics: Acid Phosphatase; Adult; Aged; Aged, 80 and over; Alkaline Phosphatase; Antineoplastic Combined Chemotherapy Protocols; beta 2-Microglobulin; Biomarkers; Biomarkers, Tumor; Bone Neoplasms; C-Reactive Protein; Cytarabine; Dexamethasone; Diphosphonates; Female; Glycoproteins; Humans; Interleukin-6; Isoenzymes; Male; Middle Aged; Multiple Myeloma; Osteocalcin; Osteoclasts; Osteogenesis; Osteoprotegerin; Pamidronate; Paraproteins; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Reference Values; Sensitivity and Specificity; Tartrate-Resistant Acid Phosphatase; Vincristine

We have been using the B9/BM1 murine bone marrow metastasis model to study the function of adhesion molecules in the cell-cell interactions and transendothelial migration, necessary for tumor metastasis. The cell surface phenotype of these cells, which colonize vertebral and femoral marrow after intravenous injection, shows great similarity to that of human myeloma cells. In the present study, we investigated the interaction between B9/BM1 cells and osteoclasts, which likely support tumor metastasis in bone marrow. We found that co-culturing B9/BM1 cells and bone marrow-derived endothelial cells (BMECs) in the presence of vitamin D3 and M-CSF promoted differentiation of primary osteoclast progenitors to osteoclasts (detected by TRAP staining), and that this effect was blocked when BMECs were separated from the other cells by a porous polycarbonate membrane. Flow cytometry analysis showed that BMECs expressed RANKL (receptor activator of NF-kappaB ligand) protein on their surface, and that this expression was up-regulated by co-culture with B9/BM1 cells. Accordingly, RT-PCR showed expression of RANKL mRNA also to be up-regulated in BMECs co-cultured with B9/BM1 cells. Addition of OPG (osteoprotegerin, a decoy RANKL receptor) to the co-culture system completely blocked osteoclast induction, as did addition of anti-CD44 antibody. Furthermore, intravenous injection of B9/BM1 cells substantially increased the numbers of TRAP-positive osteoclasts detected in mice in vivo. Taken together, these findings suggest that B9/BM1 myeloma cells act via CD44 to stimulate RANKL expression on BMECs, which in turn physically interact with osteoclast progenitors to promote their differentiation to osteoclasts and metastasis in bone marrow.

Topics: Animals; Bone Marrow Cells; Bone Marrow Neoplasms; Cell Communication; Cell Differentiation; Coculture Techniques; Endothelial Cells; Female; Glycoproteins; Interleukin-6; Mice; Multiple Myeloma; Osteoclasts; Osteoprotegerin; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Tumor Cells, Cultured

Multiple myeloma (MM) is a hematologic malignancy characterized by accumulation of plasma cells in the bone marrow (BM). Bone destruction is a complication of the disease and is usually associated with severe morbidity. The balance between receptor activator of nuclear factor-kappaB (NF-kappaB) ligand and osteoprotegerin (OPG) is of major importance in bone homeostasis. We have recently shown that serum OPG levels are lower in patients with myeloma than in healthy individuals. Here we show that myeloma cells can bind, internalize, and degrade OPG, thereby providing a possible explanation for the lower levels of OPG in the BM of patients with MM. This process is dependent on interaction of OPG with heparan sulfates on the myeloma cells. The results suggest a novel biologic mechanism for the bone disease associated with MM and that treatment of the bone disease with OPG lacking the heparin-binding domain should be considered.

Topics: Aged; Biodegradation, Environmental; Biological Transport; Biopsy; Bone Marrow Cells; Female; Glycoproteins; Humans; Immunohistochemistry; Male; Microscopy, Confocal; Multiple Myeloma; Osteoprotegerin; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Tumor Cells, Cultured

The biologic mechanisms involved in the pathogenesis of multiple myeloma (MM) bone disease are not completely understood. Recent evidence suggests that T cells may regulate bone resorption through the cross-talk between the critical osteoclastogenetic factor, receptor activator of nuclear factor-kappaB ligand (RANKL), and interferon gamma (IFN-gamma) that strongly suppresses osteoclastogenesis. Using a coculture transwell system we found that human myeloma cell lines (HMCLs) increased the expression and secretion of RANKL in activated T lymphocytes and similarly purified MM cells stimulated RANKL production in autologous T lymphocytes. In addition, either anti-interleukin 6 (anti-IL-6) or anti-IL-7 antibody inhibited HMCL-induced RANKL overexpression. Consistently, we demonstrated that HMCLs and fresh MM cells express IL-7 mRNA and secrete IL-7 in the presence of IL-6 and that bone marrow (BM) IL-7 levels were significantly higher in patients with MM. Moreover, we found that the release of IFN-gamma by T lymphocytes was reduced in presence of both HMCLs and purified MM cells. Furthermore, in a stromal cell-free system, osteoclastogenesis was stimulated by conditioned medium of T cells cocultured with HMCLs and inhibited by recombinant human osteoprotegerin (OPG; 100 ng/mL to 1 microg/mL). Finally, RANKL mRNA was up-regulated in BM T lymphocytes of MM patients with severe osteolytic lesions, suggesting that T cells could be involved at least in part in MM-induced osteolysis through the RANKL overexpression.

Topics: Aged; Bone Marrow Cells; Carrier Proteins; Coculture Techniques; Culture Media, Conditioned; Female; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Interleukin-6; Interleukin-7; Lymphocyte Activation; Male; Membrane Glycoproteins; Middle Aged; Multiple Myeloma; Neoplasm Proteins; Osteoclasts; Osteolysis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Recombinant Proteins; RNA, Messenger; RNA, Neoplasm; Stromal Cells; T-Lymphocytes

On June 26-27, 2001, the Sixth Research Roundtable in Multiple Myeloma, entitled "The Role of the Bone Microenvironment in Multiple Myeloma," was held and focused on the biology of cell-to-cell interactions, the mediators of bone disease, and novel treatment strategies for myeloma. Studies on cell-cell interactions showed that vascular cell adhesion molecule 1, expressed by local endothelial and stromal cells, binds to tumor cell surface integrins in which expression may be increased by tumor cell-derived chemokines such as macrophage inflammatory protein (MIP) 1alpha. These adhesive interactions increase production and release of vascular endothelial growth factor (VEGF). Studies on myeloma bone disease showed the ligand for receptor activator of nuclear transcription factor-kappaB (RANKL) was expressed on tumor cells and stromal cells associated with myeloma cells and was critical for osteoclast-induced osteolysis. Blockade of RANKL suppressed osteoclast maturation, bone resorption, and tumor development. Bisphosphonates, in addition to reducing osteoclast mobility and inducing osteoclast apoptosis, also decreased tumor cell adhesion to stroma. Immunomodulatory drugs such as thalidomide analogues targeted these tumor cell-stromal cell interactions, blocking both secretion of cytokines and activation of intracellular signaling pathways required for tumor survival and growth. These agents induced tumor cell apoptosis, decreased neovascularization, and potentiated natural killer cell activity. The proteasome inhibitor PS-341 also prevented expression of adhesion molecules and cytokines and triggered tumor cell apoptosis, even in drug-resistant cell lines, while showing minimal activity in healthy cells. In addition, potential therapeutic agents under investigation, which included RANKL antagonists, protein prenylation inhibitors, and osteoblast growth factors, were discussed.

Topics: Adjuvants, Immunologic; Bone and Bones; Bone Marrow; Bone Resorption; Boronic Acids; Bortezomib; Carrier Proteins; Cell Division; Chemokine CCL3; Chemokine CCL4; Diphosphonates; Endothelial Growth Factors; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Lymphokines; Macrophage Inflammatory Proteins; Membrane Glycoproteins; Multiple Myeloma; Osteoclasts; Osteolysis; Osteoprotegerin; Protease Inhibitors; Protein Prenylation; Pyrazines; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The new members of the tumour necrosis factor (TNF) receptor-ligand family, receptor activator of nuclear factor-kappaB ligand (RANKL) and its receptor RANK, play a crucial role in osteoclast differentiation and activation. An increased expression of RANKL and/or RANK may be involved in the excessive bone resorption observed in multiple myeloma (MM). We used immunohistochemistry to study RANK and RANKL expression in bone marrow (BM) biopsies obtained at diagnosis in 15 MM patients, six patients with monoclonal gammopathy of undetermined significance (MGUS) and 10 normal BM biopsies. Plasma cells were not labelled with anti-RANKL or anti-RANK antibodies. In all biopsies, RANKL was expressed in endosteal bone surface, around vessels and in cells characterized by cytoplasmic expansions. These last cells did not express CD45 and were vimentin positive, corresponding to bone marrow stromal cells. Numerous stromal cells expressed RANKL in MM and MGUS specimens, with a greater expression in MM than in MGUS. Very few cells were stained with anti-RANKL in normal BM specimens. With the anti-RANK antibody, small mononuclear cells in the bone microenvironment were positive and were identified as erythroblast cells. In conclusion, we showed that RANKL was expressed in reticular stromal cells, with a greater intensity in myeloma specimens. These results suggest that RANKL overexpressed by bone marrow stromal cells may contribute to the high rate of bone resorption observed in MM.

Topics: Bone Marrow Cells; Carrier Proteins; Case-Control Studies; Glycoproteins; Humans; Immunohistochemistry; Membrane Glycoproteins; Multiple Myeloma; Osteoprotegerin; Paraproteinemias; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Stromal Cells

Human apolipoprotein E4 (apoE4) binds preferentially to lower density lipoproteins, including very low density lipoproteins, and is associated with increased risk of atherosclerosis and neurodegenerative disorders, including Alzheimer's disease. This binding preference is the result of the presence of Arg-112, which causes Arg-61 in the amino-terminal domain to interact with Glu-255 in the carboxyl-terminal domain. ApoE2 and apoE3, which have Cys-112, bind preferentially to high density lipoproteins (HDL) and do not display apoE4 domain interaction. Mouse apoE, like apoE4, contains the equivalent of Arg-112 and Glu-255, but lacks the critical Arg-61 equivalent (it contains Thr-61). Thus, mouse apoE does not display apoE4 domain interaction and, as a result, behaves like human apoE3, including preferential binding to HDL. To assess the potential role of apoE4 domain interaction in atherosclerosis and neurodegeneration, we sought to introduce apoE4 domain interaction into mouse apoE. Replacing Thr-61 in mouse apoE with arginine converted the binding preference from HDL to very low density lipoproteins in vitro, suggesting that apoE4 domain interaction could be introduced into mouse apoE in vivo. Using gene targeting in embryonic stem cells, we created mice expressing Arg-61 apoE. Heterozygous Arg-61/wild-type apoE mice displayed two phenotypes found in human apoE4/E3 heterozygotes: preferential binding to lower density lipoproteins and reduced abundance of Arg-61 apoE in the plasma, reflecting its more rapid catabolism. These findings demonstrate the successful introduction of apoE4 domain interaction into mouse apoE in vivo. The Arg-61 apoE mouse model will allow the effects of apoE4 domain interaction in lipoprotein metabolism, atherosclerosis, and neurodegeneration to be determined.

Topics: Animals; Apolipoprotein E4; Apolipoproteins E; Bone Marrow; Carrier Proteins; Disease Progression; Gene Expression Regulation; Glycoproteins; Humans; Immunohistochemistry; In Situ Hybridization; Membrane Glycoproteins; Mice; Mice, SCID; Multiple Myeloma; Neoplasm Transplantation; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Transplantation, Heterologous; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Osteoprotegerin (OPG), the neutralizing decoy receptor for the osteoclast activator RANK ligand, was measured in serum taken from patients with multiple myeloma at the time of diagnosis. Median OPG was lower in the patients with myeloma (7.4 ng/mL; range, 2.6-80; n = 225) than in healthy age- and sex-matched controls (9.0 ng/mL; range 5.1-130; n = 40; P =.02). Importantly, OPG levels were associated with degree of radiographically assessed skeletal destruction (P =.01). The median OPG level in patients lacking osteolytic lesions was 9.1 ng/mL, as compared with 7.6 ng/mL and 7.0 ng/mL, respectively, in patients with minor or advanced osteolytic disease. Furthermore, OPG levels were associated with World Health Organization performance status (P =.003) and correlated to serum levels of carboxy-terminal propeptide of type I procollagen (PICP; P <.001) but not with clinical stage or survival. These findings suggest impaired OPG function in myeloma and give a rationale for OPG as a therapeutic agent against myeloma bone disease.

Topics: Adult; Aged; Aged, 80 and over; Case-Control Studies; Glycoproteins; Health Status Indicators; Humans; Middle Aged; Multiple Myeloma; Osteolysis; Osteoprotegerin; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Although osteolysis is a common complication in patients with multiple myeloma (MM), the biologic mechanisms involved in the pathogenesis of MM-induced bone disease are poorly understood. Two factors produced by stromal-osteoblastic cells seem critical to the regulation of bone resorption: osteoprotegerin (OPG) and its ligand (OPGL). OPGL stimulates osteoclast differentiation and activity, whereas OPG inhibits these processes. The present study investigated whether myeloma cells affect physiologic OPG/OPGL balance in the bone marrow (BM) environment. Ten human myeloma cell lines and myeloma cells isolated from 26 consecutive patients with MM failed to express OPGL and only rarely produced a low amount of OPG. In a coculture system, human myeloma cells up-regulated OPGL expression but strongly down-regulated OPG production in preosteoblastic (preOB) or stromal cells (BMSCs) of primary human BM at the mRNA and protein levels. This effect, which was dependent on cell-to-cell contact between myeloma cells and BMSCs or preOB, partially involved the integrin VLA-4. In addition, overexpression of OPGL mRNA occurred in ex vivo BM cultures obtained from MM patients as compared with healthy donors, and immunohistochemical staining performed on BM biopsy specimens showed an increase of OPGL and a reduction of OPG expression in MM patients as compared with healthy subjects. In summary, these data indicate that myeloma cells affect the OPG/OPGL ratio in the BM environment and tend to confirm that the OPG/OPGL system is involved in the pathogenesis of MM-induced bone disease.

Topics: Aged; Aged, 80 and over; Bone Marrow; Carrier Proteins; Coculture Techniques; Female; Gene Expression; Glycoproteins; Humans; Immunohistochemistry; Male; Membrane Glycoproteins; Middle Aged; Multiple Myeloma; Osteoblasts; Osteoclasts; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; RNA, Messenger; Stromal Cells; Tumor Cells, Cultured

Multiple myeloma is a B-cell malignancy characterized by the accumulation of plasma cells in the bone marrow and the development of osteolytic bone disease. The present study demonstrates that myeloma cells express the critical osteoclastogenic factor RANKL (the ligand for receptor activator of NF-kappa B). Injection of 5T2MM myeloma cells into C57BL/KaLwRij mice resulted in the development of bone disease characterized by a significant decrease in cancellous bone volume in the tibial and femoral metaphyses, an increase in osteoclast formation, and radiologic evidence of osteolytic bone lesions. Dual-energy x-ray absorptiometry demonstrated a decrease in bone mineral density (BMD) at each of these sites. Treatment of mice with established myeloma with recombinant osteoprotegerin (OPG) protein, the soluble decoy receptor for RANKL, prevented the development of lytic bone lesions. OPG treatment was associated with preservation of cancellous bone volume and inhibition of osteoclast formation. OPG also promoted an increase in femoral, tibial, and vertebral BMD. These data suggest that the RANKL/RANK/OPG system may play a critical role in the development of osteolytic bone disease in multiple myeloma and that targeting this system may have therapeutic potential.

Topics: Animals; Bone and Bones; Bone Density; Carrier Proteins; Flow Cytometry; Gene Expression; Glycoproteins; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Multiple Myeloma; Neoplasm Transplantation; Osteolysis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Syndecan-1 (CD138) is a heparan sulfate-bearing proteoglycan present on the surface of myeloma cells where it mediates myeloma cell-cell and cell-extracellular matrix adhesion. In this study, we examined myeloma cell lines for cell membrane localization of syndecan-1. On some cells we note a striking localization of syndecan-1 to a single small membrane protrusion, with the remainder of the cell surface being mostly negative for syndecan-1. Examination of cell morphology reveals that a proportion of cells from myeloma cell lines, as well as primary myeloma cells, are polarized, with a uropod on one end and lamellipodia on the other end. On these polarized cells, syndecan-1 is specifically targeted to the uropod, but in contrast, on nonpolarized cells syndecan-1 is evenly distributed over the entire cell surface. In addition to syndecan-1, several other cell surface molecules localize specifically to the uropod, including CD44 and CD54. Functional assays reveal that myeloma cell lines with a high proportion of polarized cells have a much higher migratory potential than cell lines with few polarized cells. Moreover, the uropod is the cell pole preferentially involved in aggregation of myeloma cells and in adhesion of myeloma cells to osteoblast-like cells. When polarized myeloma cells are incubated with heparin-binding proteins, like hepatocyte growth factor or osteoprotegerin, they concentrate in the uropod. These data indicate that syndecan-1 is targeted to the uropod of polarized myeloma cells and that this targeting plays a role in promoting cell-cell adhesion and may also regulate the biological activity of heparin-binding cytokines.

Topics: Animals; Antibodies, Monoclonal; Carrier Proteins; Cell Adhesion; Cell Membrane; Cell Movement; Cell Polarity; Epithelial Cells; Glycoproteins; Hepatocyte Growth Factor; Humans; Hyaluronan Receptors; Intercellular Adhesion Molecule-1; LDL-Receptor Related Protein-Associated Protein; Membrane Glycoproteins; Mice; Mice, SCID; Multiple Myeloma; Osteoprotegerin; Proteoglycans; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Syndecan-1; Syndecans; Tumor Cells, Cultured