Tumor-supportive and Osteoclastogenic Changes Induced by Breast Cancer-derived Factors Are Reversed by Inhibition of γ-Secretase

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

A cannabinoid 2 receptor agonist attenuates bone cancer-induced pain and bone loss

AimsCannabinoid CB₂ agonists have been shown to alleviate behavioral signs of inflammatory and neuropathic pain in animal models. AM1241, a CB₂ agonist, does not demonstrate central nervous system side effects seen with CB₁ agonists such as hypothermia and catalepsy. Metastatic bone cancer causes severe pain in patients and is treated with analgesics such as opiates. Recent reports suggest that sustained opiates can produce paradoxical hyperalgesic actions and enhance bone destruction in a murine model of bone cancer. In contrast, CB₂ selective agonists have been shown to reduce bone loss associated with a model of osteoporosis. Here we tested whether a CB₂ agonist administered over a 7 day period inhibits bone cancer-induced pain as well as attenuates cancer-induced bone degradation.Main methodsA murine bone cancer model was used in which osteolytic sarcoma cells were injected into the intramedullary space of the distal end of the femur. Behavioral and radiographic image analysis was performed at days 7, 10 and 14 after injection of tumor cells into the femur.Key findingsOsteolytic sarcoma within the femur produced spontaneous and touch evoked behavioral signs of pain within the tumor-bearing limb. The systemic administration of AM1241 acutely or for 7 days significantly attenuated spontaneous and evoked pain in the inoculated limb. Sustained AM1241 significantly reduced bone loss and decreased the incidence of cancer-induced bone fractures.SignificanceThese findings suggest a novel therapy for cancer-induced bone pain, bone loss and bone fracture while lacking many unwanted side effects seen with current treatments for bone cancer pain.     

Inhibition of p38-MAPK signaling pathway attenuates breast cancer induced bone pain and disease progression in a murine model of cancer-induced bone pain

Background

Mechanisms driving cancer-induced bone pain are poorly understood. A central factor implicated to be a key player in the process of tumorigenesis, osteoclastogenesis and nociception is p38 MAPK. We determined the role of p38 MAPK in a mouse model of breast cancer induced bone pain in which mixed osteolytic and osteoblastic remodeling occurs.

Results

In cancer-treated mice, acute as well as chronic inhibition of p38 MAPK with SB203580 blocked flinching and guarding behaviors in a dose-dependent manner whereas no effect on thresholds to tactile stimuli was observed. Radiographic analyses of bones demonstrated that chronic inhibition of p38 MAPK reduced bone loss and incidence of spontaneous fracture in cancer-treated mice. Histological analysis of bones collected from mice treated with the p38 MAPK inhibitor showed complete absence of osteoblastic growth in the intramedullary space as well as significantly reduced tumor burden.

Conclusions

Blockade of non-evoked pain behaviors but not hypersensitivity suggests differences in the underlying mechanisms of specific components of the pain syndrome and a possibility to individualize aspects of pain management. While it is not known whether the role of p38 MAPK signaling can be expanded to other cancers, the data suggest a need for understanding molecular mechanisms and cellular events that initiate and maintain cancer-induced bone pain for effective management for both ongoing pain as well as breakthrough pain.     

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.     

Advances in understanding bone cancer pain

Experimental animal models of bone cancer pain have emerged and findings have provided a unique glimpse into unraveling the mechanism that drives this debilitating condition. Key contributors to the generation and maintenance of bone cancer pain are tumor-induced osteolysis, tumor itself, and production of nociceptive mediators in the bone-tumor microenvironment.     

Human interleukin-1-induced murine osteoclastogenesis is dependent on RANKL, but independent of TNF-alpha

Although interleukin-1 (IL-1) has been implicated in the pathogenesis of inflammatory osteolysis, the means by which it recruits osteoclasts and promotes bone destruction are largely unknown. Recently, a cytokine-driven, stromal cell-free mouse osteoclastogenesis model was established. A combination of macrophage colony stimulating factor (M-CSF) and receptor activator of NFkappaB ligand (RANKL) was proven to be sufficient in inducing differentiation of bone marrow hematopoietic precursor cells to bone-resorbing osteoclasts in the absence of stromal cells or osteoblasts. This study utilizes this model to examine the impact of human IL-1beta on in vitro osteoclastogenesis of bone marrow progenitor cells. We found that osteoclast precursor cells failed to undergo osteoclastogenesis when treated with IL-1 alone. In contrast, IL-1 dramatically up-regulated osteoclastogenesis by 2.5- to 4-folds in the presence of RANKL and M-CSF. The effect can be significantly blocked by IL-1 receptor antagonist (p < 0.01). Tumor necrosis factor-alpha (TNF-alpha) was undetectable in the culture medium of differentiating osteoclasts induced by IL-1. Adding exogenous TNF-alpha neutralizing antibody had no influence on the IL-1-induced effect as well. These results show that in the absence of stromal cells, IL-1 exacerbates osteoclastogenesis by cooperating with RANKL and M-CSF, while TNF-alpha is not involved in this IL-1-stimulated osteoclast differentiation pathway.     

Paclitaxel inhibits osteoclast formation and bone resorption via influencing mitotic cell cycle arrest and RANKL-induced activation of NF-κB and ERK

Pathological bone destruction (osteolysis) is a hallmark of many bone diseases including tumor metastasis to bone, locally osteolytic giant cell tumor (GCT) of bone, and Paget's disease. Paclitaxel is frequently prescribed in the treatment of several malignant tumors where it has been shown to exert beneficial effects on bone lesions. However, the mechanism(s) through which paclitaxel regulates osteoclast formation and function remain ill defined. In the present study, we demonstrate that paclitaxel dose-dependently inhibits receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis in both RAW264.7 cells and mouse bone marrow macrophage (BMM) systems. In addition, paclitaxel treatment reduces the bone resorptive activity of human osteoclasts derived from GCT of bone, and attenuates lipopolysaccharide (LPS)-induced osteolysis in a mouse calvarial model. Complementary cellular and biochemical analyses revealed that paclitaxel induces mitotic arrest of osteoclastic precursor cells. Furthermore, luciferase reporter gene assays and western blot analysis indicate that paclitaxel modulates key RANKL-induced activation pathways that are essential to osteoclast formation including NF-κB and ERK. Collectively, our findings demonstrate a role for paclitaxel in the regulation of osteoclast formation and function and uncover potential mechanism(s) through which paclitaxel alleviates pathological osteolysis.     

Osteoblasts suppress high bone turnover caused by osteolytic breast cancer in-vitro

The skeleton is the most common site of breast cancer metastasis, which can occur in up to 85% of patients during their lifetime. The morbidity associated with bone metastases in patients with breast cancer includes pathological fractures, bone pain, hypercalcaemia, and spinal cord compression. When breast cancer metastasizes to bone, the balance of bone resorption (mediated by osteoclasts) and bone formation (mediated by osteoblasts) favors bone resorption, which leads to net bone destruction (i.e., osteolysis). Anti-resorptive agents such as bisphosphonates are commonly used to treat bone resorption in osteoporosis or osteolytic cancer patients. However, bisphosphonates by themselves are unable to rebuild lost bone tissue, and can cause severe side effects. In this study, we developed a bovine bone explant culture system and have observed that murine osteoblasts can modulate the activity of osteotropic human breast cancer cells on this substrate. Using markers of bone metabolism, we observe diminished bone turnover in organ culture following the addition of exogenous osteoblasts. The data presented in this study supports further investigation into the use of cytotherapies to limit breast cancer mediated osteolysis.     

Daphnetin attenuates LPS-induced osteolysis and RANKL mediated osteoclastogenesis through suppression of ERK and NFATc1 pathways

Aseptic prosthetic loosening and periprosthetic infection resulting in inflammatory osteolysis is a leading complication of total joint arthroplasty (TJA). Excessive bone destruction around the bone and prosthesis interface plays a key role in the loosening prostheses leading to revision surgery. The bacterial endotoxins or implant-derived wear particles-induced inflammatory response is the major cause of the elevated osteoclast formation and activity. Thus, agents or compounds that can attenuate the inflammatory response and/or inhibit the elevated osteoclastogenesis and excessive bone resorption would provide a promising therapeutic avenue to prevent aseptic prosthetic loosening in TJA. Daphnetin (DAP), a natural coumarin derivative, is clinically used in Traditional Chinese Medicine for the treatment of rheumatoid arthritis due to its anti-inflammatory properties. In this study, we report for the first time that DAP could protect against lipopolysaccharide-induced inflammatory bone destruction in a murine calvarial osteolysis model in vivo. This protective effect of DAP can in part be attributed to its direct inhibitory effect on RANKL-induced osteoclast differentiation, fusion, and bone resorption in vitro. Biochemical analysis found that DAP inhibited the activation of the ERK and NFATc1 signaling cascades. Collectively, our findings suggest that DAP as a natural compound has potential for the treatment of inflammatory osteolysis.     

Interactions between cancer and bone marrow cells induce osteoclast differentiation factor expression and osteoclast-like cell formation in vitro

Cancer cells metastasized to bone induce osteoclastogenesis for bone destruction. Coculture of either mouse melanoma B16 or breast cancer Balb/c-MC cells with mouse bone marrow cells (BMCs) induced osteoclast-like cells, which were not observed when cancer cells were segregated from BMCs. Osteoclast differentiation factor (ODF), also known as receptor activator of NF-kappaB ligand (RANKL), is a direct mediator of many osteotropic factors. Neither BMCs, B16 nor Balb/c-MC cells alone expressed ODF mRNA. However, coculture of these cancer cells with BMCs induced ODF expression, which was prevented by indomethacin. Moreover, the coculture with cancer cells inhibited secretion of osteoprotegerin/osteoclastogenesis inhibitory factor (OPG/OCIF), an inhibitory decoy receptor for ODF, from BMCs. Thus, enhanced osteoclastogenesis in the presence of cancer cells might be due to an increase in ODF activity. These results suggest that interactions between cancer cells and BMCs induce ODF expression and suppress OPG/OCIF level in metastatic foci resulting in pathological osteoclastogenesis for bone destruction.     

Prostate cancer bone metastases promote both osteolytic and osteoblastic activity

Advanced prostate cancer is frequently accompanied by the development of metastasis to bone. In the past, prostate cancer bone metastases were characterized as being osteoblastic (i.e., increasing bone density) based on radiographs. However, emerging evidence suggests that development of prostate cancer bone metastases requires osteoclastic activity in addition to osteoblastic activity. The complexities of how prostate tumor cells influence bone remodeling are just beginning to be elucidated. Prostate cancer cells produce a variety of pro-osteoblastic factors that promote bone mineralization. For example, both bone morphogenetic proteins and endothelin-1 have well recognized pro-osteoblastic activities and are produced by prostate cancer cells. In addition to factors that enhance bone mineralization prostate cancer cells produced factors that promote osteoclast activity. Perhaps the most critical pro-osteoclastogenic factor produced by prostate cancer cells is receptor activator of NFkappaB ligand (RANKL), which has been shown to be required for the development of osteoclasts. Blocking RANKL results in inhibiting prostate cancer-induced osteoclastogenesis and inhibits development and progression of prostate tumor growth in bone. These findings suggest that targeting osteoclast activity may be of therapeutic benefit. However, it remains to be defined how prostate cancer cells synchronize the combination of osteoclastic and osteoblastic activity. We propose that as the bone microenvironment is changed by the developing cancer, this in turn influences the prostate cancer cells' balance between pro-osteoclastic and pro-osteoblastic activity. Accordingly, the determination of how the prostate cancer cells and bone microenvironment crosstalk are important to elucidate how prostate cancer cells modulate bone remodeling.     

Curcumin analogue UBS109 prevents bone loss in breast cancer bone metastasis mouse model: involvement in osteoblastogenesis and osteoclastogenesis

Bone metastasis of breast cancer typically leads to osteolysis, which causes severe pathological bone fractures and hypercalcemia. Bone homeostasis is skillfully regulated through osteoblasts and osteoclasts. Bone loss with bone metastasis of breast cancer may be due to both activation of osteoclastic bone resorption and suppression of osteoblastic bone formation. This study was undertaken to determine whether the novel curcumin analogue UBS109 has preventive effects on bone loss induced by breast cancer cell bone metastasis. Nude mice were inoculated with breast cancer MDA-MB-231 bone metastatic cells (10⁶ cells/mouse) into the head of the right and left tibia. One week after inoculation, the mice were treated with control (vehicle), oral administration (p.o.) of UBS109 (50 or 150 mg/kg body weight), or intraperitoneal administration (i.p.) of UBS109 (10 or 20 mg/kg body weight) once daily for 5 days per week for 7 weeks. After UBS109 administration for 7 weeks, hind limbs were assessed using an X-ray diagnosis system and hematoxylin and eosion staining to determine osteolytic destruction. Bone marrow cells obtained from the femurs and tibias were cultured to estimate osteoblastic mineralization and osteoclastogenesis ex vivo and in vitro. Remarkable bone loss was demonstrated in the tibias of mice inoculated with breast cancer MDA-MB-231 bone metastatic cells. This bone loss was prevented by p.o. administration of UBS109 (50 and 150 mg/kg body weight) and i.p. treatment of UBS109 (10 and 20 mg/kg) in vivo. Culture of bone marrow cells obtained from the bone tissues of mice with breast cancer cell bone metastasis showed suppressed osteoblastic mineralization and stimulated osteoclastogenesis ex vivo. These changes were not seen after culture of the bone marrow cells obtained from mice treated with UBS109. Moreover, UBS109 was found to stimulate osteoblastic mineralization and suppress lipopolysaccharide (LPS)-induced osteoclastogenesis in bone marrow cells obtained from normal nude mice in vitro. These findings suggest that the novel curcumin analogue UBS109 prevents breast cancer cell bone metastasis-induced bone loss by stimulating osteoblastic mineralization and suppressing osteoclastogenesis.     

Functional Upregulation of Nav1.8 Sodium Channels on the Membrane of Dorsal Root Ganglia Neurons Contributes to the Development of Cancer-Induced Bone Pain

We have previously reported that enhanced excitability of dorsal root ganglia (DRG) neurons contributes to the development of bone cancer pain, which severely decreases the quality of life of cancer patients. Nav1.8, a tetrodotoxin-resistant (TTX-R) sodium channel, contributes most of the sodium current underlying the action potential upstroke and accounts for most of the current in later spikes in a train. We speculate that the Nav1.8 sodium channel is a potential candidate responsible for the enhanced excitability of DRG neurons in rats with bone cancer pain. Here, using electrophysiology, Western blot and behavior assays, we documented that the current density of TTX-R sodium channels, especially the Nav1.8 channel, increased significantly in DRG neurons of rats with cancer-induced bone pain. This increase may be due to an increased expression of Nav1.8 on the membrane of DRG neurons. Accordantly, blockade of Nav1.8 sodium channels by its selective blocker A-803467 significantly alleviated the cancer-induced mechanical allodynia and thermal hyperalgesia in rats. Taken together, these results suggest that functional upregulation of Nav1.8 channels on the membrane of DRG neurons contributes to the development of cancer-induced bone pain.     

Scutellarein inhibits RANKL-induced osteoclast formation in vitro and prevents LPS-induced bone loss in vivo

Osteoporosis, arthritis, Peget's disease, bone tumor, periprosthetic joint infection, and periprosthetic loosening have a common characteristic of osteolysis, which is characterized by the enhanced osteoclastic bone resorptive function. At present, the treatment target of these diseases is to interfere with osteoclastic formation and function. Scutellarein (Scu), a flavonoids compound, can inhibit the progress of tumor and inflammation. However, the role of Scu in inflammatory osteolysis isn’t elucidated clearly. Our study showed that Scu inhibited bone destruction induced by LPS in vivo and OC morphology and function induced by RANKL in vitro. Mechanistic studies revealed that Scu suppressed osteoclastic marker gene expression by RANKL-induced, such as Ctsk9, Mmp9, Acp5, and Atp6v0d2. In addition, we found that the inhibition effects of osteoclastogenesis and bone resorption function of Scu were mediated via attenuating NF-κB and NFAT signaling pathways. In conclusion, the results showed that Scu may become a potential new drug for the treatment of inflammatory osteolysis.     

Osteoprotegerin blocks bone cancer-induced skeletal destruction, skeletal pain and pain-related neurochemical reorganization of the spinal cord

Bone cancer pain is common among cancer patients and can have a devastating effect on their quality of life. A chief problem in designing new therapies for bone cancer pain is that it is unclear what mechanisms drive this distinct pain condition. Here we show that osteoprotegerin, a secreted 'decoy' receptor that inhibits osteoclast activity, also blocks behaviors indicative of pain in mice with bone cancer. A substantial part of the actions of osteoprotegerin seems to result from inhibition of tumor-induced bone destruction that in turn inhibits the neurochemical changes in the spinal cord that are thought to be involved in the generation and maintenance of cancer pain. These results demonstrate that excessive tumor-induced bone destruction is involved in the generation of bone cancer pain and that osteoprotegerin may provide an effective treatment for this common human condition.     

Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo

Wear particle‐stimulated inflammatory bone destruction and the consequent aseptic loosening remain the primary causes of artificial prosthesis failure and revision. Previous studies have demonstrated that curcumin has a protective effect on bone disorders and inflammatory diseases and can ameliorate polymethylmethacrylate‐induced osteolysis in vivo. However, the effect on immunomodulation and the definitive mechanism by which curcumin reduces the receptor activators of nuclear factor‐kappa B ligand (RANKL)‐stimulated osteoclast formation and prevents the activation of osteoclastic signalling pathways are unclear. In this work, the immunomodulation effect and anti‐osteoclastogenesis capacities exerted by curcumin on titanium nanoparticle‐stimulated macrophage polarization and on RANKL‐mediated osteoclast activation and differentiation in osteoclastic precursor cells in vitro were investigated. As expected, curcumin inhibited RANKL‐stimulated osteoclast maturation and formation and had an immunomodulatory effect on macrophage polarization in vitro. Furthermore, studies aimed to identify the potential molecular and cellular mechanisms revealed that this protective effect of curcumin on osteoclastogenesis occurred through the amelioration of the activation of Akt/NF‐κB/NFATc1 pathways. Additionally, an in vivo mouse calvarial bone destruction model further confirmed that curcumin ameliorated the severity of titanium nanoparticle‐stimulated bone loss and destruction. Our results conclusively indicated that curcumin, a major biologic component of Curcuma longa with anti‐inflammatory and immunomodulatory properties, may serve as a potential therapeutic agent for osteoclastic diseases.