Plumbagin inhibits breast tumor bone metastasis and osteolysis by modulating the tumor-bone microenvironment

Bone metastasis is a common and serious consequence of breast cancer. Bidirectional interaction between tumor cells and the bone marrow microenvironment drives a so-called 'vicious cycle' that promotes tumor cell malignancy and stimulates osteolysis. Targeting these interactions and pathways in the tumor-bone microenvironment has been an encouraging strategy for bone metastasis therapy. In the present study, we examined the effects of plumbagin on breast cancer bone metastasis. Our data indicated that plumbagin inhibited cancer cell migration and invasion, suppressed the expression of osteoclast-activating factors, altered the cancer cell induced RANKL/OPG ratio in osteoblasts, and blocked both cancer cell- and RANKL-stimulated osteoclastogenesis. In mouse model of bone metastasis, we further demonstrated that plumbagin significantly repressed breast cancer cell metastasis and osteolysis, inhibited cancer cell induced-osteoclastogenesis and the secretion of osteoclast-activating factors in vivo. At the molecular level, we found that plumbagin abrogated RANKL-induced NF-κB and MAPK pathways by blocking RANK association with TRAF6 in osteoclastogenesis, and by inhibiting the expression of osteoclast-activating factors through the suppression of NF-κB activity in breast cancer cells. Taken together, our data demonstrate that plumbagin inhibits breast tumor bone metastasis and osteolysis by modulating the tumor-bone microenvironment and that plumbagin may serve as a novel agent in the treatment of tumor bone metastasis.     

OPG/RANKL/RANK系统与骨破坏性疾病

近年来发现的OPG/RANKL/RANK系统在破骨细胞生成中起着至关重要的作用,是骨骼生理研究领域的重大进展。成骨细胞、骨髓基质细胞、激活的T淋巴细胞表达RANKL,与破骨细胞前体细胞或成熟破骨细胞表面上的RANK结合后,促进破骨细胞的分化及骨吸收活性。成骨细胞及骨髓基质细胞分泌表达OPG可与RANKL竞争性结合,从而阻断RANKL与RANK之间的相互作用。体内多种激素或因子通过影响骨髓微环境内的OPG/RANKL比率来调节骨代谢。此外,乳腺上皮细胞表达有RANK,孕期在性激素的诱导下可表达RANKL,OPG/RANKL/RANK系统在孕期乳腺发育以及母体向胎儿的钙转运过程中发挥重要作用。阻断RANKL/RANK通路有望给骨质疏松、类风湿关节炎及癌症骨转移等骨破坏性疾病的治疗开辟新的途径。进一步研究应了解OPG/RANKL/RANK系统与其它信号传导途径的关系,重视骨骼、免疫及内分泌系统之间的相互作用。目前,开发与OPG功能相似或促进其表达的合成药物有可能成为具有良好经济效益和社会效益的产业。     

前列腺癌骨转移：肿瘤细胞与骨微环境之间的相互作用

  肿瘤转移包括一系列复杂的过程,主要涉及肿瘤细胞由原发部位脱离开始,到肿瘤细胞在其它转移部位——比如骨骼,生长增殖的多个关键性步骤.“种子和土壤学说”预示骨微环境中表达许多因子,吸引多种肿瘤细胞的迁移以及促进肿瘤的增殖.通过肿瘤细胞与其所处生长环境中的双向和动态的作用,促进肿瘤在骨骼中的发展.因此,骨微环境中产生的因子对肿瘤骨转移具有重要意义.本综述以前列腺癌为例,总结了肿瘤转移的机理研究概况,特别强调了目前有关肿瘤细胞与骨微环境之间相互作用研究的重要性,并提出了未来的研究方向     

A novel receptor activator of NF-kappaB (RANK) cytoplasmic motif plays an essential role in osteoclastogenesis by committing macrophages to the osteoclast lineage

Receptor activator of NF-kappaB (RANK) ligand (RANKL) and its receptor RANK play an essential role in osteoclastogenesis and osteoclast function by activating several signaling pathways. However, several lines of evidence suggest that RANK also transmits an unidentified signaling pathway(s) essential for osteoclastogenesis. To identify the novel pathway(s), we carried out a detailed structure/function study of the RANK cytoplasmic domain. A series of studies using numerous deletion/point mutants elucidated a specific 4-amino acid motif (535IVVY538) essential for osteoclastogenesis. This novel motif plays a crucial role in committing macrophages to the osteoclast lineage but is not implicated in osteoclast function or survival. Moreover, this motif does not activate the known RANK signaling pathways, indicating that it initiates a novel pathway(s). The identification of the novel motif not only provides critical insight into RANK signaling in osteoclastogenesis, but more importantly, the RANK motif and its downstream signaling pathways may represent specific therapeutic targets for various bone diseases, including postmenopausal osteoporosis.     

The “bone morphogenic proteins” pathways in bone and joint diseases: Translational perspectives from physiopathology to therapeutic targets

A large body of evidence supports an important role of bone morphogenic proteins (BMPs) pathways in skeletal development in the embryo. BMPs are also involved in skeletal homeostasis and diseases in the adult. They were first identified as major bone anabolic agents and recent advances indicate that they also regulate osteoclastogenesis and joint components via multiple cross-talks with other signaling pathways. This review attempts to integrate these data in the pathogenesis of bone and joints diseases, such as osteoporosis, fracture healing, osteoarthritis, inflammatory arthritis, or bone metastasis. The use of recombinant BMPs in bone tissue engineering and in the treatment of skeletal diseases, or future therapeutic strategies targeting BMPs signal and its regulators, will be discussed based on these considerations.     

A RANK/TRAF6-dependent signal transduction pathway is essential for osteoclast cytoskeletal organization and resorptive function

Signaling through receptor activator of nuclear factor-kappaB (RANK) is essential for the differentiation and activation of osteoclasts, the cell principally responsible for bone resorption. Animals genetically deficient in RANK or the cognate RANK ligand are profoundly osteopetrotic because of the lack of bone resorption and remodeling. RANK provokes biochemical signaling via the recruitment of intracellular tumor necrosis factor receptor-associated factors (TRAFs) after ligand binding and receptor oligomerization. To understand the RANK-mediated signal transduction mechanism in osteoclastogenesis, we have designed a system to recapitulate osteoclast differentiation and activation in vitro by transfer of the RANK cDNA into hematopoietic precursors genetically deficient in RANK. Gene transfer of RANK constructs that are selectively incapable of binding different TRAF proteins revealed that TRAF pathways downstream of RANK that affect osteoclast differentiation are functionally redundant. In contrast, the interaction of RANK with TRAF6 is absolutely required for the proper formation of cytoskeletal structures and functional resorptive activity of osteoclasts. Moreover, signaling via the interleukin-1 receptor, which also utilizes TRAF6, rescues the osteoclast activation defects observed in the absence of RANK/TRAF6 interactions. These studies are the first to define the functional domains of the RANK cytoplasmic tail that control specific differentiation and activation pathways in osteoclasts.     

TGF—β在前列腺癌骨转移中的研究进展

进展期前列腺癌多会发生骨转移,导致患者骨质破坏甚至死亡。前列腺癌发生骨转移的机制目前尚未研究清楚。既往多认为是因为前列腺癌细胞表面携带者容易在骨环境中生长的表型。但是目前多认为是肿瘤细胞与骨骼微环境之间的相互作用导致的结果。它们之间是通过细胞因子来传递信息。在众多因子中,TGF-β对前列腺癌骨转移灶中的各种细胞都起着重要作用。研究表明在体外实验中TGF-β的作用极易受到细胞生长环境的影响,表现出不同的功能。这提示着TGF-β信号通路和其他信号通路之间存在非常强的交互作用。本文的重点在于对TGF-β在前列腺癌骨转移中的作用研究进展进行综述,阐述TGF-β对转移灶中不同细胞的作用．为今后肿瘤的治疗研究寻找一个好的方向。     

TGF-beta在前列腺癌骨转移中的研究进展

进展期前列腺癌多会发生骨转移,导致患者骨质破坏甚至死亡。前列腺癌发生骨转移的机制目前尚未研究清楚。既往多认 为是因为前列腺癌细胞表面携带者容易在骨环境中生长的表型。但是目前多认为是肿瘤细胞与骨骼微环境之间的相互作用导致的结果。它们之间是通过细胞因子来传递信息。在众多因子中,TGF-beta对前列腺癌骨转移灶中的各种细胞都起着重要作用。研究表明在体外实验中TGF-beta的作用极易受到细胞生长环境的影响,表现出不同的功能。这提示着TGF-beta信号通路和其他信号通路之间存在非常强的交互作用。本文的重点在于对TGF-beta在前列腺癌骨转移中的作用研究进展进行综述,阐述TGF-beta对转移灶中不同细胞的作用,为今后肿瘤的治疗研究寻找一个好的方向。     

Osteoporosis: the current status of mesenchymal stem cell-based therapy

Osteoporosis, or bone loss, is a progressive, systemic skeletal disease that affects millions of people worldwide. Osteoporosis is generally age related, and it is underdiagnosed because it remains asymptomatic for several years until the development of fractures that confine daily life activities, particularly in elderly people. Most patients with osteoporotic fractures become bedridden and are in a life-threatening state. The consequences of fracture can be devastating, leading to substantial morbidity and mortality of the patients. The normal physiologic process of bone remodeling involves a balance between bone resorption and bone formation during early adulthood. In osteoporosis, this process becomes imbalanced, resulting in gradual losses of bone mass and density due to enhanced bone resorption and/or inadequate bone formation. Several growth factors underlying age-related osteoporosis and their signaling pathways have been identified, such as osteoprotegerin (OPG)/receptor activator of nuclear factor B (RANK)/RANK ligand (RANKL), bone morphogenetic protein (BMP), wingless-type MMTV integration site family (Wnt) proteins and signaling through parathyroid hormone receptors. In addition, the pathogenesis of osteoporosis has been connected to genetics. The current treatment of osteoporosis predominantly consists of antiresorptive and anabolic agents; however, the serious adverse effects of using these drugs are of concern. Cell-based replacement therapy via the use of mesenchymal stem cells (MSCs) may become one of the strategies for osteoporosis treatment in the future.     

Hypoxia and TGF-β Drive Breast Cancer Bone Metastases through Parallel Signaling Pathways in Tumor Cells and the Bone Microenvironment

Background

Most patients with advanced breast cancer develop bone metastases, which cause pain, hypercalcemia, fractures, nerve compression and paralysis. Chemotherapy causes further bone loss, and bone-specific treatments are only palliative. Multiple tumor-secreted factors act on the bone microenvironment to drive a feed-forward cycle of tumor growth. Effective treatment requires inhibiting upstream regulators of groups of prometastatic factors. Two central regulators are hypoxia and transforming growth factor (TGF)- β. We asked whether hypoxia (via HIF-1α) and TGF-β signaling promote bone metastases independently or synergistically, and we tested molecular versus pharmacological inhibition strategies in an animal model.

Methodology/Principal Findings

We analyzed interactions between HIF-1α and TGF-β pathways in MDA-MB-231 breast cancer cells. Only vascular endothelial growth factor (VEGF) and the CXC chemokine receptor 4 (CXCR4), of 16 genes tested, were additively increased by both TGF-β and hypoxia, with effects on the proximal promoters. We inhibited HIF-1α and TGF-β pathways in tumor cells by shRNA and dominant negative receptor approaches. Inhibition of either pathway decreased bone metastasis, with no further effect of double blockade. We tested pharmacologic inhibitors of the pathways, which target both the tumor and the bone microenvironment. Unlike molecular blockade, combined drug treatment decreased bone metastases more than either alone, with effects on bone to decrease osteoclastic bone resorption and increase osteoblast activity, in addition to actions on tumor cells.

Conclusions/Significance

Hypoxia and TGF-β signaling in parallel drive tumor bone metastases and regulate a common set of tumor genes. In contrast, small molecule inhibitors, by acting on both tumor cells and the bone microenvironment, additively decrease tumor burden, while improving skeletal quality. Our studies suggest that inhibitors of HIF-1α and TGF-β may improve treatment of bone metastases and increase survival.     

Expression pattern of receptor activator of NFκB (RANK) in a series of primary solid tumors and related bone metastases

Receptor activator of NFκB ligand (RANKL), RANK, and osteoprotegerin (OPG) represent the key regulators of bone metabolism both in normal and pathological conditions, including bone metastases. To our knowledge, no previous studies investigated and compared RANK expression in primary tumors and in bone metastases from the same patient. We retrospectively examined RANK expression by immunohistochemistry in 74 bone metastases tissues from solid tumors, mostly breast, colorectal, renal, lung, and prostate cancer. For 40 cases, tissue from the corresponding primary tumor was also analyzed. Sixty‐six (89%) of the 74 bone metastases were RANK‐positive and, among these, 40 (59.5%) showed more than 50% of positive tumor cells. The median percentage of RANK‐positive cells was 60% in primary tumors and metastases, without any statistically significant difference between the two groups (P = 0.194). The same percentage was obtained by considering only cases with availability of samples both from primary and metastasis. Our study shows that RANK is expressed by solid tumors, with high concordance between bone metastasis and corresponding primary tumor. These data highlight the central role of RANK/RANKL/OPG pathway as potential therapeutic target not only in bone metastasis management, but also in the adjuvant setting. J. Cell. Physiol. 226: 780–784, 2011. © 2010 Wiley‐Liss, Inc.     

Guaiacol suppresses osteoclastogenesis by blocking interactions of RANK with TRAF6 and C‐Src and inhibiting NF‐κB,MAPK and AKT pathways

Angelica sinensis (AS; Dang Gui), a traditional Chinese herb, has for centuries been used for the treatment of bone diseases, including osteoporosis and osteonecrosis. However, the effective ingredient and underlying mechanisms remain elusive. Here, we identified guaiacol as the active component of AS by two‐dimensional cell membrane chromatography/C18 column/time‐of‐flight mass spectrometry (2D CMC/C18 column/TOFMS). Guaiacol suppressed osteoclastogenesis and osteoclast function in bone marrow monocytes (BMMCs) and RAW264.7 cells in vitro in a dose‐dependent manner. Co‐immunoprecipitation indicated that guaiacol blocked RANK‐TRAF6 association and RANK‐C‐Src association. Moreover, guaiacol prevented phosphorylation of p65, p50, IκB (NF‐κB pathway), ERK, JNK, c‐fos, p38 (MAPK pathway) and Akt (AKT pathway), and reduced the expression levels of Cathepsin K, CTR, MMP‐9 and TRAP. Guaiacol also suppressed the expression of nuclear factor of activated T‐cells cytoplasmic 1(NFATc1) and the RANKL‐induced Ca²⁺ oscillation. In vivo, it ameliorated ovariectomy‐induced bone loss by suppressing excessive osteoclastogenesis. Taken together, our findings suggest that guaiacol inhibits RANKL‐induced osteoclastogenesis by blocking the interactions of RANK with TRAF6 and C‐Src, and by suppressing the NF‐κB, MAPK and AKT signalling pathways. Therefore, this compound shows therapeutic potential for osteoclastogenesis‐related bone diseases, including postmenopausal osteoporosis.     

Therapeutic implications of osteoprotegerin

Osteoprotegerin (OPG), a member of the tumor necrosis factor (TNF) receptor superfamily, contributes determinatively to the bone remodeling as well as to the pathogenetic mechanism of bone malignancies and disorders of mineral metabolism. There is additional evidence that OPG can promote cell survival by inhibiting TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. A number of recent in vitro, in vivo and clinical studies have defined the role of the RANK/RANKL/OPG pathway in skeletal and vascular diseases. These works were the milestone of the deep understanding of the mechanism of OPG. This review provides an overview of the potential innovative therapeutic strategies of OPG in metastatic breast and prostate carcinoma, multiple myeloma, postmenopausal osteoporosis, glucocorticoid-induced osteoporosis and rheumatoid arthritis. Special reference is given to the increasing evidence that RANKL and OPG may link the skeletal with the vascular system.     

Prostate cancer cells and bone stromal cells mutually interact with each other through bone morphogenetic protein-mediated signals

Functional interactions between cancer cells and the bone microenvironment contribute to the development of bone metastasis. Although the bone metastasis of prostate cancer is characterized by increased ossification, the molecular mechanisms involved in this process are not fully understood. Here, the roles of bone morphogenetic proteins (BMPs) in the interactions between prostate cancer cells and bone stromal cells were investigated. In human prostate cancer LNCaP cells, BMP-4 induced the production of Sonic hedgehog (SHH) through a Smad-dependent pathway. In mouse stromal MC3T3-E1 cells, SHH up-regulated the expression of activin receptor IIB (ActR-IIB) and Smad1, which in turn enhanced BMP-responsive reporter activities in these cells. The combined stimulation with BMP-4 and SHH of MC3T3-E1 cells cooperatively induced the expression of osteoblastic markers, including alkaline phosphatase, bone sialoprotein, collagen type II α1, and osteocalcin. When MC3T3-E1 cells and LNCaP cells were co-cultured, the osteoblastic differentiation of MC3T3-E1 cells, which was induced by BMP-4, was accelerated by SHH from LNCaP cells. Furthermore, LNCaP cells and BMP-4 cooperatively induced the production of growth factors, including fibroblast growth factor (FGF)-2 and epidermal growth factor (EGF) in MC3T3-E1 cells, and these may promote the proliferation of LNCaP cells. Taken together, our findings suggest that BMPs provide favorable circumstances for the survival of prostate cancer cells and the differentiation of bone stromal cells in the bone microenvironment, possibly leading to the osteoblastic metastasis of prostate cancer.     

Matricellular proteins as regulators of cancer metastasis to bone

Metastasis is the major cause of death in cancer patients, and a frequent site of metastasis for many cancers is the bone marrow. Therefore, understanding the mechanisms underlying the metastatic process is necessary for future prevention and treatment. The tumor microenvironment is now known to play a role in the metastatic cascade, both at the primary tumor and in metastatic sites, and includes both cellular and non-cellular components. The extracellular matrix (ECM) provides structural support and signaling cues to cells. One particular group of molecules associated with the ECM, known as matricellular proteins, modulate multiple aspects of tumor biology, including growth, migration, invasion, angiogenesis and metastasis. These proteins are also important for normal function in the bone by regulating bone formation and bone resorption. Recent studies have described a link between some of these proteins and metastasis of various tumors to the bone. The aim of this review is to summarize what is currently known about matricellular protein influence on bone metastasis. Particular attention to the contribution of both tumor cells and non-malignant cells in the bone has been given.     

Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment

The identity of cells that establish the hematopoietic microenvironment (HME) in human bone marrow (BM), and of clonogenic skeletal progenitors found in BM stroma, has long remained elusive. We show that MCAM/CD146-expressing, subendothelial cells in human BM stroma are capable of transferring, upon transplantation, the HME to heterotopic sites, coincident with the establishment of identical subendothelial cells within a miniature bone organ. Establishment of subendothelial stromal cells in developing heterotopic BM in vivo occurs via specific, dynamic interactions with developing sinusoids. Subendothelial stromal cells residing on the sinusoidal wall are major producers of Angiopoietin-1 (a pivotal molecule of the HSC "niche" involved in vascular remodeling). Our data reveal the functional relationships between establishment of the HME in vivo, establishment of skeletal progenitors in BM sinusoids, and angiogenesis.     

Protein Expression Profiling of Giant Cell Tumors of Bone Treated with Denosumab

Giant cell tumors of bone (GCTB) are locally aggressive osteolytic bone tumors. Recently, some clinical trials have shown that denosumab is a novel and effective therapeutic option for aggressive and recurrent GCTB. This study was performed to investigate the molecular mechanism underlying the therapeutic effect of denosumab. Comparative proteomic analyses were performed using GCTB samples which were taken before and after denosumab treatment. Each expression profile was analyzed using the software program to further understand the affected biological network. One of identified proteins was further evaluated by gelatin zymography and an immunohistochemical analysis. We identified 13 consistently upregulated proteins and 19 consistently downregulated proteins in the pre- and post-denosumab samples. Using these profiles, the software program identified molecular interactions between the differentially expressed proteins that were indirectly involved in the RANK/RANKL pathway and in several non-canonical subpathways including the Matrix metalloproteinase pathway. The data analysis also suggested that the identified proteins play a critical functional role in the osteolytic process of GCTB. Among the most downregulated proteins, the activity of MMP-9 was significantly decreased in the denosumab-treated samples, although the residual stromal cells were found to express MMP-9 by an immunohistochemical analysis. The expression level of MMP-9 in the primary GCTB samples was not correlated with any clinicopathological factors, including patient outcomes. Although the replacement of tumors by fibro-osseous tissue or the diminishment of osteoclast-like giant cells have been shown as therapeutic effects of denosumab, the residual tumor after denosumab treatment, which is composed of only stromal cells, might be capable of causing bone destruction; thus the therapeutic application of denosumab would be still necessary for these lesions. We believe that the protein expression patterns and the results of the network analysis will provide a better understanding of the effects of denosumab administration in patients with GCTB.     

Activation of the RalGEF/Ral pathway promotes prostate cancer metastasis to bone

A hallmark of metastasis is organ specificity; however, little is known about the underlying signaling pathways responsible for the colonization and growth of tumor cells in target organs. Since tyrosine kinase receptor activation is frequently associated with prostate cancer progression, we have investigated the role of a common signaling intermediary, activated Ras, in prostate cancer metastasis. Three effector pathways downstream of Ras, Raf/extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase, and Ral guanine nucleotide exchange factors (RalGEFs), were assayed for their ability to promote the metastasis of a tumorigenic, nonmetastatic human prostate cancer cell line, DU145. Oncogenic Ras promoted the metastasis of DU145 to multiple organs, including bone and brain. Activation of the Raf/ERK pathway stimulated metastatic colonization of the brain, while activation of the RalGEF pathway led to bone metastases, the most common organ site for prostate cancer metastasis. In addition, loss of RalA in the metastatic PC3 cell line inhibited bone metastasis but did not affect subcutaneous tumor growth. Loss of Ral appeared to suppress expansive growth of prostate cancer cells in bone, whereas homing and initial colonization were less affected. These data extend our understanding of the functional roles of the Ral pathway and begin to identify signaling pathways relevant for organ-specific metastasis.