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Introduction: Cancer-induced bone pain (CIBP) is one of the most common and debilitating complications associated with bone metastasis. Although our understanding of the precise mechanism is limited, it has been known that bone is densely innervated, and that CIBP is elicited as a consequence of increased neurogenesis, reprogramming, and axonogenesis in conjunction with sensitization and excitation of sensory nerves (SNs) in response to the noxious stimuli that are derived from the tumor microenvironment developed in bone. Recent studies have shown that the sensitized and excited nerves innervating the tumor establish intimate communications with cancer cells by releasing various tumor-stimulating factors for tumor progression., Approaches: In this review, the role of the interactions of cancer cells and SNs in bone in the pathophysiology of CIBP will be discussed with a special focus on the role of the noxious acidic tumor microenvironment, considering that bone is in nature hypoxic, which facilitates the generation of acidic conditions by cancer. Subsequently, the role of SNs in the regulation of cancer progression in the bone will be discussed together with our recent experimental findings., Conclusion: It is suggested that SNs may be a newly-recognized important component of the bone microenvironment that contribute to not only in the pathophysiology of CIBP but also cancer progression in bone and dissemination from bone. Suppression of the activity of bone-innervating SNs, thus, may provide unique opportunities in the treatment of cancer progression and dissemination, as well as CIBP., (© 2023. The Japanese Society Bone and Mineral Research.)

Breast cancer (BC) bone metastasis causes bone pain (BP), which detrimentally damages the quality of life and outcome of patients with BC. However, the mechanism of BC‑BP is poorly understood, and effective treatments are limited. The present study demonstrated a novel mechanism of BC‑BP using a mouse model of bone pain, in which mouse (EO771) and human (MDA‑MB‑231) BC cells were injected in the bone marrow cavity of tibiae. Western blot analysis using sensory nerves, in vivo assessment of cancer pain and in vitro calcium flux analysis were performed. These mice developed progressive BC‑BP in tibiae in conjunction with an upregulation of phosphorylated pERK1/2 and cAMP‑response element‑binding protein (pCREB), which are molecular indicators of neuron excitation, in the dorsal root ganglia (DRG) of sensory nerves. Importantly, mice injected with BC cells, in which the expression of the lactic acid transporter monocarboxylate transporter 4 (MCT4) was silenced, exhibited decreased BC‑BP with downregulated expression of pERK1/2 and pCREB in the DRG and reduced circulating levels of lactate compared with mice injected with parental BC cells. Further, silencing of the cell‑surface orphan receptor for lactate, G protein‑coupled receptor 81 (GPR81), in the F11 sensory neuron cells decreased lactate‑promoted upregulation of pERK1/2 and Ca 2+ influx, suggesting that the sensory neuron excitation was inhibited. These results suggested that lactate released from BC cells via MCT4 induced BC‑BP through the activation of GPR81 of sensory neurons. In conclusion, the activation of GPR81 of sensory neurons by lactate released via MCT4 from BC was demonstrated to contribute to the induction of BC‑BP, and disruption of the interactions among lactate, MCT4 and GPR81 may be a novel approach to control BC‑BP.

Purpose of Review: Sensory nerves (SNs) richly innervate bone and are a component of bone microenvironment. Cancer metastasis in bone, which is under the control of the crosstalk with bone microenvironment, induces bone pain via excitation of SNs innervating bone. However, little is known whether excited SNs in turn affect bone metastasis., Recent Findings: Cancer cells colonizing bone promote neo-neurogenesis of SNs and excite SNs via activation of the acid-sensing nociceptors by creating pathological acidosis in bone, evoking bone pain. Denervation of SNs or inhibition of SN excitation decreases bone pain and cancer progression and increases survival in preclinical models. Importantly, patients with cancers with increased SN innervation complain of cancer pain and show poor outcome. SNs establish the crosstalk with cancer cells to contribute to bone pain and cancer progression in bone. Blockade of SN excitation may have not only analgesic effects on bone pain but also anti-cancer actions on bone metastases.

Background: Giant cell tumor of bone (GCTB) is a rare primary bone tumor, characterized by osteoclast-like giant cells that express receptor activator of nuclear factor-kappa B (RANK), and stromal cells that express RANK ligand (RANKL), a key mediator of osteoclast activation. A RANKL-specific inhibitor, denosumab, was predicted to reduce osteolysis and control disease progression in patients with GCTB., Patients and Methods: Seventeen patients with GCTB were enrolled. Patients were treated with denosumab at 120 mg every 4 weeks, with a loading dose of 120 mg on days 8 and 15. To evaluate efficacy, objective tumor response was evaluated prospectively by an independent imaging facility on the basis of prespecified criteria., Results: The proportion of patients with an objective tumor response was 88% based on best response using any tumor response criteria. The proportion of patients with an objective tumor response using individual response criteria was 35% based on the modified Response Evaluation Criteria in Solid Tumors (RECIST) criteria, 82% based on the modified European Organization for Research and Treatment of Cancer (EORTC) criteria, and 71% based on inverse Choi criteria. The median time of study treatment was 13.1 months., Conclusion: The findings demonstrate that denosumab has robust clinical efficacy in the treatment of GCTB., (© The Author 2015. Published by Oxford University Press on behalf of the European Society for Medical Oncology.)

Solid and hematologic cancer colonized bone produces a number of pathologies. One of the most common complications is bone pain. Cancer-associated bone pain (CABP) is a major cause of increased morbidity and diminishes the quality of life and affects survival. Current treatments do not satisfactorily control CABP and can elicit adverse effects. Thus, new therapeutic interventions are needed to manage CABP. However, the mechanisms responsible for CABP are poorly understood. The observation that specific osteoclast inhibitors can reduce CABP in patients indicates a critical role of osteoclasts in the pathophysiology of CABP. Osteoclasts create an acidic extracellular microenvironment by secretion of protons via vacuolar proton pumps during bone resorption. In addition, bone-colonized cancer cells also release protons and lactate via plasma membrane pH regulators to avoid intracellular acidification resulting from increased aerobic glycolysis known as the Warburg effect. Since acidosis is algogenic for sensory neurons and bone is densely innervated by sensory neurons that express acid-sensing nociceptors, the acidic bone microenvironments can evoke CABP. Understanding of the mechanism by which the acidic extracellular microenvironment is created in cancer-colonized bone and the expression and function of the acid-sensing nociceptors are regulated should facilitate the development of novel approaches for management of CABP. Here, the contribution of the acidic microenvironment created in cancer-colonized bone to elicitation of CABP and potential therapeutic implications of blocking the development and recognition of acidic microenvironment will be described. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Bone is one of the most preferential metastatic target sites for cancers. The biological crosstalk between metastatic cancer cells and bone microenvironment is critical to the pathophysiology of bone metastases. For example, It is well established that PTH-rP production in cancer cells stimulated by bone-derived TGF-β facilitates bone metastasis through promoting bone resorption by osteoclasts, thereby establishing "vicious cycle" between metastatic cancer cells and bone. In addition, recent studies identified several new players including platelets and MDSCs which contribute to the development of bone metastasis. In this review, we will overview the current topics on the mechanism by which bone metastasis is modulated at cellular and molecular levels.

Solid cancers such as breast, prostate and lung cancer have a predilection for spreading to bone. Accumulating data suggest that the crosstalk between metastatic cancer cells and bone-resorbing osteoclasts plays a central role in the development and progression of bone metastases. Recent studies have revealed that osteoblasts mediate this crosstalk by expressing the receptor activator of NF-kappaB ligand (RANKL) in response to cancer-produced osteotropic factors. The RANKL promotes osteoclast formation and bone resorption via the binding to RANK expressed in hematopoietic osteoclast precursor cells and mature osteoclasts. Denosumab is the humanized anti-RANKL neutralizing monoclonal antibody and thus would be a specific and effective therapeutic agent for the treatment of bone metastases. In fact, clinical studies clearly demonstrated that denosumab significantly inhibited the development of skeletal-related events associated with bone metastases and indicate its usefulness for bone metastases.

The continuous release of bone-stored growth factors after bone resorption promotes the colonization of circulating cancer cells. However, the precise role of each of the various growth factors remains unclear. In this study, we investigated the role of bone-derived insulin-like growth factor (IGF) in the development of bone metastases in an animal model of breast cancer. We found that local stimulation of calvarial bone resorption before cell inoculation stimulated subsequent bone metastases to that site in vivo, although inhibition of bone resorption inhibited bone metastases. Anchorage-independent growth of cancer cells was stimulated by the culture supernatants from resorbed bones, which contained elevated levels of IGF-I. This stimulation was blocked by IGF type I receptor (IGF-IR) neutralizing antibody, but not antibody targeting other bone-stored growth factors including TGF-β, fibroblast growth factors, and platelet-derived growth factors. Although recombinant human IGF-I caused IGF-IR tyrosine autophosphorylation, followed by activation of Akt and NF-κB in cancer cells, dominant-negative inhibition of IGF-IR, Akt, or NF-κB significantly reduced bone metastases with increased apoptosis and decreased mitosis in metastatic cells. Together, our findings suggest that bone-derived IGF-I bridges the crosstalk between bone and metastasized cancer cells via activation of the IGF-IR/Akt/NF-κB pathway. Disruption of this pathway therefore may represent a promising therapeutic intervention for bone metastasis., (©2012 AACR.)

Bone metastasis is relatively common in patients with breast, prostate, and lung cancer, and may cause severe bone pain, pathological fractures, hypercalcemia and other bone-related complications that drastically undermine quality of life. The currently-available treatments for bone metastasis are unsatisfactory in their effectiveness and outcome. Bone continuously undergoes remodeling through a repeated cycle of osteoclastic bone resorption and osteoblastic bone formation. When cancer cells metastasize to the bone, their growth is promoted under the influence of a variety of growth factors that are supplied from the bone as a consequence of osteoclastic bone resorption. This, in turn, causes an increased production of osteoclast- and osteoblast-stimulating cytokines in these cancer cells, leading to a further increase in bone remodeling. This vicious cycle between bone and metastatic cancer cells supports the development and progression of bone metastases. Accumulating evidence shows that RANKL, a cytokine expressed in osteoblasts/bone marrow stromal cells, plays an important role in the formation, activation, and survival of osteoclasts, which are key players in bone remodeling. RANKL is thus expected to inhibit bone metastasis by disrupting this vicious cycle, and is considered a rational new therapeutic intervention for bone metastasis. In animal models, inhibition with RANKL has been shown to prevent the development of bone metastases. Based on the promising results from these preclinical studies, a fully human monoclonal anti-RANKL antibody, denosumab, has been developed. In three phase III clinical trials of denosumab versus zoledronic acid(ZA)in advanced cancer patients with bone metastases, denosumab was superior or equal to ZA in delaying the time to the first on-study skeletal-related events(SRE). In conclusion, denosumab, a fully human monoclonal anti-RANKL antibody with a targeted mechanism of action, enables us to more easily and effectively treat bone metastases and manage cancer patients with bone metastases.

Breast cancer has a predilection for spreading to bone. Accumulating data suggest that the interactions between breast cancer cells and bone-resorbing osteoclasts mediated initially by bone-derived growth factors and consequently by RANKL in osteoblasts and RANK in pre-osteoclasts are critical to the development and progression of bone metastases. In addition, recent studies have revealed that breast cancer cells strongly express RANK and thereby can be directly influenced by osteoblast-derived RANKL. These results warrant the use of the humanized anti-RANKL neutralizing antibody (denosumab) as a specific and more effective therapeutic agent for the treatment of bone metastasis in breast cancer patients.

Accumulating evidence indicates that the acidic microenvironments critically influence malignant behaviors of cancer including invasiveness, metastasis, and chemoresistance. Because the vacuolar-type H(+)-ATPase (V-ATPase) has been shown to cause extracellular acidification by pumping protons, we studied the role of V-ATPase in distant metastasis. Real-time PCR analysis revealed that the high-metastatic B16-F10 melanoma cells strongly expressed the a3 isoform V-ATPase compared to the low-metastatic B16 parental cells. Consistent with this, B16-F10 cells created acidic environments in lung metastases by acridine orange staining and strong a3 V-ATPase expression in bone metastases by immunohistochemistry. Immunocytochemical analysis showed B16-F10 cells expressed a3 V-ATPase not only in cytoplasm but also plasma membrane, whereas B16 parental cells exhibited its expression only in cytoplasm. Of note, knockdown of a3 V-ATPase suppressed invasiveness and migration with reduced MMP-2 and MMP-9 expression in B16-F10 cells and significantly decreased lung and bone metastases, despite that tumor growth was not altered. Importantly, administration of a specific V-ATPase a3 inhibitor FR167356 reduced bone metastasis of B16-F10 cells. These results suggest that a3 V-ATPase promotes distant metastasis of B16-F10 cells by creating acidic environments via proton secretion. Our results also suggest that inhibition of the development of cancer-associated acidic environments by suppressing a3 V-ATPase could be a novel therapeutic approach for the treatment of cancer metastasis., (©2011 AACR.)

Bone, as well as the lung and liver, is among the sites of predilection for cancer metastasis. The bone stores large amounts of growth factors such as insulin-like growth factors and transforming growth factor-b, and provides fertile soil for metastatic cancer cells by continuously releasing these bone-stored growth factors, which are a consequence of osteoclastic bone resorption. Metastatic cancer cells in turn produce osteoclast-stimulating cytokines such as parathyroid hormone-related protein( PTH-rP), prostaglandin E2.(PGE2), and various interleukins(ILs). These cancer-produced osteoclast-stimulating cytokines bind to their cognitive receptors and promote the expression of ligands for the receptor activators of nuclear factor kB (RANKL)in osteoblasts. RANKL then binds to its receptor RANK, expressed in pre-osteoclasts, stimulates mature osteoclast formation, and subsequently, osteoclastic bone resorption. This vicious cycle between metastatic cancer cells and osteoclasts is critical to the development and progression of bone metastases. In addition, it is likely that metastatic cancer cells are influenced by bone environments(or niche)and acquire additional capacities such as an epithelial-mesenchymal transition(EMT), allowing them to be resistant to chemotherapy or apoptosis, to survive in a dormant state, or to aggressively spread to distant organs including lung and liver. Thus, the bone can serve as transit port. Disrupting this cycle by inhibiting osteoclastic bone resorption, antagonizing bone-derived growth factors, and neutralizing RANKL or PTH-rP, should be a promising therapeutic intervention for bone metastases. Bisphosphonates(BP)are specific inhibitors of osteoclasts, and have been shown to significantly reduce skeletal-related events(SRE)associated with bone metastasis. Denosumab is a neutralizing monoclonal antibody to RANKL and has recently been found to inhibit SRE more effectively than BP. Further understanding of the crosstalk communication between metastatic cancer cells and bone at the molecular level should lead us to design novel, more effective and specific treatments for cancer patients with bone metastases.

Substantial data from clinical trials and epidemiological studies show promising results for use of statins in many cancers, including mammary carcinoma. Breast tumor primarily metastasizes to bone to form osteolytic lesions, causing severe pain and pathological fracture. Here, we report that simvastatin acts as an inhibitor of osteolysis in a mouse model of breast cancer skeletal metastasis of human mammary cancer cell MDA-MB-231, which expresses the mutant p53R280K. Simvastatin and lovastatin attenuated migration and invasion of MDA-MB-231 and BT-20 breast tumor cells in culture. Acquisition of phenotype to express the cancer stem cell marker, CD44, leads to invasive potential of the tumor cells. Interestingly, statins significantly decreased the expression of CD44 protein via a transcriptional mechanism. shRNA-mediated down-regulation of CD44 markedly reduced the migration and invasion of breast cancer cells in culture. We identified that in the MDA-MB-231 cells, simvastatin elevated the levels of mutated p53R280K, which was remarkably active as a transcription factor. shRNA-derived inhibition of mutant p53R280K augmented the expression of CD44, leading to increased migration and invasion. Finally, we demonstrate an inverse correlation between expression of p53 and CD44 in the tumors of mice that received simvastatin. Our results reveal a unique function of statins, which foster enhanced expression of mutant p53R280K to prevent breast cancer cell metastasis to bone.

Bone pain is the most common complications in bone metastases, causing increased morbidity and undermining quality of life (QOL) in patients. It has been considered that algesic factors produced by tumor tissues and nerve injury are involved in pain progression. However, the molecular mechanisms of bone pain are still complex and not fully understood. Recent studies show that acidic microenvironment created in bone metastasis is relevant to pain signal through the activation of acid-sensing nociceptor in sensory neurons. These elucidations might be lead to the development of therapeutic approaches for cancer pain.

Bone is one of the most preferential metastatic target sites for cancers. However, based on the anatomical structure of the vascular system, bone is not recognized as a preferential metastatic target. Therefore, the biological crosstalk between metastatic cancer cells and bone is critical to the development and progression of bone metastases. Bone microenvironments possess unique biological features characterized by abundant growth factors and diverse cellular network including osteoblasts, osteoclasts and hematopietic cells. Cancers develop bone metastases by utilizing these unique bone environments for colonization and bone destruction. Better understandings of precise molecular mechanisms underlying cancer and bone crosstalk would contribute to the development of new therapeutic approaches for the treatment of bone metastasis at molecular levels.

Bone pain is one of the most common complications in cancer patients with bone metastases. Although the mechanism of cancer-associated bone pain is poorly understood, clinical observations that inhibitors of osteoclasts such as bisphosphonates (BPs) efficiently reduce bone pain suggest a potential role of osteoclasts, which play a central role in the development and progression of bone metastasis. Osteoclasts dissolve bone minerals by releasing protons through the a3 isoform of the vacuolar-H(+)-ATPase, creating acidic microenvironments. In addition, cancer cells, inflammatory cells and immune cells that reside in bone metastases also produce acidic conditions by releasing protons. It has been well-known that acidic conditions due to proton release cause pain. Our study showed that the sensory nociceptive neurons innervate bone and these neurons express acid-sensing nociceptors such as the acid-sensing ion channels and transient receptor potential channel-vanilloid subfamily members. Acid signals received by these nociceptors subsequently activate intracellular signaling pathways and transcription factors in sensory neurons. The understanding of the nociceptive events following proton release and subsequent creation of acidic microenvironments leads us to design novel molecular-based approaches for reducing bone pain associated with cancer and inflammation., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The data derived from epidemiological and animal models confirm a beneficial effect of fish oil (rich in ω-3 polyunsaturated fatty acids) in the amelioration of tumor growth and progression, including breast cancer. The breast cancer patients often develop bone metastasis evidenced by osteolytic lesions, leading to severe pain and bone fracture. Using a mouse model of MDA-MB-231 human breast cancer cell metastasis to bone, here we show that fish oil diet enriched in DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid) prevents the formation of osteolytic lesions in bone, indicating suppression of cancer cell metastasis to bone. These results are supported by our data showing both DHA and EPA significantly attenuate the migration/invasion of MDA-MB-231 breast cancer cells in culture. The mechanism that limits breast cancer cells to selective metastasis to bone remains hitherto unexplored. Aberrant increased expression of CD44 is associated with generation of cancer stem cells, which contribute to metastasis of breast cancer cells. We demonstrate that DHA and EPA significantly inhibit the expression of CD44 protein and mRNA by a transcriptional mechanism. Furthermore, we show markedly reduced levels of CD44 mRNA and protein in the tumors of mice, which were fed fish oil diet than those in control diet. Our data provide the first evidence for a salutary effect of fish oil on breast cancer metastasis to bone. Our results identify a novel function of the fish oil active components, DHA and EPA, which target the cell-intrinsic pro-metastatic molecule CD44 to inhibit migration/invasion., (Published by Elsevier Inc.)

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

Cell adhesion molecules have been implicated in the selective colonization of cancer in distant organs. Breast cancer has a strong predilection for spreading to bone. Cadherin-11, which is one of the classical type-2 cadherin family members and mediates homophilic cell-cell adhesion, is constitutively expressed in stromal and osteoblastic cells in bone marrow. Elevated cadherin-11 expression is also found in aggressive human breast cancers. Here, we investigated the role of the interactions between breast cancer cells and bone marrow stromal/osteoblastic cells via cadherin-11 in the selective spread to bone. The bone-seeking clone of the MDA-MB-231 human breast cancer cells showed greater cadherin-11 expression than the parental and the brain-seeking clone. Cadherin-11 overexpression in MDA-MB-231 cells increased bone metastases with promoted bone resorption, while the natural variant form of cadherin-11 that is unable to establish cell-cell adhesion did not. Of note, introduction of cadherin-11 showed no effects on lung metastases. Fluorescence-activated cell sorter analysis using the fluorescent dye-labeled cancer cells showed that early colonization in bone marrow was increased by cadherin-11. Co-cultures with the MC3T3-E1 osteoblastic cells that constitutively expressed cadherin-11 caused an up-regulation of parathyroid hormone-related protein (PTH-rP) production in MDA-MB-231 cells overexpressing cadherin-11. The conditioned medium of the co-cultures increased osteoclastogenesis, which was blocked by a neutralizing antibody to PTH-rP. In conclusion, our results suggest that cadherin-11 promotes homing and migration to bone and osteoclastogenesis through mediating the homophilic interactions of breast cancer cells with marrow stromal/osteoblastic cells, thereby enhancing bone metastases.

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

Hypoxia is a common feature of solid tumors and is associated with their malignant phenotype. The transcription factor hypoxia-inducible factor-1 (HIF-1) is a major regulator of adaptation to hypoxia and is implicated in the malignant progression of cancers. Here, we studied whether hypoxia and HIF-1 expression contribute to the development of bone metastases using a well-characterized animal model of bone metastasis in MDA-MB-231 human breast cancer cells. To study the role of hypoxia in bone metastases, we tested the effects of the fusion protein (TOP3), the oxygen-dependent degradation domain of HIF-1alpha fused with HIV-TAT, and procaspase-3. TOP3 selectively induced apoptosis in hypoxic tumor cells in vitro and significantly reduced bone metastases in vivo. We next examined the role of HIF-1 in bone metastases by establishing MDA-MB-231 cells overexpressing constitutively active or dominant-negative HIF-1alpha (MDA/CA-HIF or MDA/DN-HIF, respectively). Bone metastases of MDA/CA-HIF were significantly increased with elevated number of CD31-positive blood vessels. In contrast, bone metastases were significantly reduced in MDA/DN-HIF. Because the progression of osteolytic bone metastases is due in part to the imbalance between bone formation and bone resorption, we examined the effects of hypoxia and HIF-1 on the differentiation of osteoblasts and osteoclasts. Hypoxia and CA-HIF overexpression markedly inhibited osteoblastic differentiation, whereas hypoxia increased osteoclast-like cell formation. In conclusion, these results suggest that tumor-associated hypoxia and HIF-1 expression promote the progression of bone metastases in breast cancer. Our results also suggest that hypoxia and HIF-1 lead to the development of osteolytic bone metastases by suppressing osteoblast differentiation and promoting osteoclastogenesis.

Bone pain is one of the most common complications in cancer patients with bone metastases. Previous findings that inhibitors of osteoclastic bone resorption such as bisphosphonates (BPs) reduce bone pain suggest a critical role of osteoclasts. Osteoclasts destroy bone by secreting protons, thereby making adjacent microenvironment acidic. Because acidosis is a well-known cause of pain, it is plausible that an osteoclasts-created acidic microenvironment may cause bone pain associated with cancer colonization in bone. To test this notion, we studied an animal model in which inoculation of MRMT-1 rat breast cancer cells into the tibiae in female rats induced hyperalgesia. Radiographic and histological analyses demonstrated that MRMT-1 cells caused aggressive bone destruction with an increased number of osteoclasts. Behavioral analyses showed that rats exhibited hyperalgesia in the tumor-inoculated legs. The BP zoledronic acid (ZOL) significantly reduced the hyperalgesia. In addition, immunohistochemical examinations revealed that c-Fos expression in the ipsilateral spinal cord neurons was increased. ZOL decreased these c-Fos-positive neurons. To investigate the role of acidosis, mRNA expression of acid-sensing receptors including acid-sensing channels (ASICs) and transient receptor potential channel-vanilloid subfamily member 1 (TRPV1) in the dorsal root ganglions (DRGs) was determined. The expression of ASIC1a and ASIC1b was increased in the ipsilateral DRGs, whereas the ASIC3 and TRPV1 expression was not changed. Of note, ZOL reduced the expression of ASIC1a and ASIC1b. In conclusion, our data suggest that an acidic microenvironment created by osteoclasts, at least in part, contributes to the induction of hyperalgesia through upregulating ASICs expression.

Matrix Metalloproteinases (MMPs) play a crucial role in breast cancer metastasis. We examined the mRNA and protein expression of several MMPs in brain- and bone-seeking clones of MDA-MB-231 breast cancer cells, their transcriptional regulation and their functional role in the metastatic process. MMP mRNA expression was examined using real-time reverse transcription polymerase chain reaction. Protein expression was examined using enzyme linked immunosorbent essay (ELISA). The inducibility of mRNA and protein expression was tested with TPA (phorbol 12-myristate 13-acetate; 50 microM); epidermal growth factor and transforming growth factor beta (20 ng/ml both). Migration and invasion assays were performed with the QCM 96-Well Migration/Invasion Assay (8 microm; Chemicon) over 24 h with or without specific MMPs inhibitors (MMP Inhibitor I Mix (5 microM); MMP-2/MMP-9 Inhibitor III (50 microM); EMD Biosciences). We found significantly higher mRNA expression of MMP-1 and -9 in brain-seeking 231-clones in comparison to -bone and -parental cells. In contrast, the mRNA expression of MMP-3 and -14 was comparable in all cells lines examined and MMP-13 expression was lower in both selective metastatic lines. MMP-2 and -8 were not expressed. ELISA revealed a higher amount of total as well as active MMP-1 and -9 in brain-seeking cells. TPA stimulation showed that MMP-1 and -9 transcription was inducible on the mRNA and protein level in 231-parental but not in 231-brain or -bone. 231-brain showed the highest migration and invasive capacity which could be decreased by the application of MMP-1 and/or MMP-9 inhibitor. Our results indicate functional importance of MMP-1 and -9 overexpression in brain metastasis in an in vitro model.

In bone metastatic lesions, osteoclasts play a key role in the development of osteolysis. Previous studies have shown that macrophage colony-stimulating factor (M-CSF) is important for the differentiation of osteoclasts. In this study, we investigated whether an inhibitor of M-CSF receptor (c-Fms) suppresses osteoclast-dependent osteolysis in bone metastatic lesions. We developed small molecule inhibitors against ligand-dependent phosphorylation of c-Fms and examined the effects of these compounds on osteolytic bone destruction in a bone metastasis model. We discovered a novel quinoline-urea derivative, Ki20227 (N-{4-[(6,7-dimethoxy-4-quinolyl)oxy]-2-methoxyphenyl}-N'-[1-(1,3-thiazole-2-yl)ethyl]urea), which is a c-Fms tyrosine kinase inhibitor. The IC(50)s of Ki20227 to inhibit c-Fms, vascular endothelial growth factor receptor-2 (KDR), stem cell factor receptor (c-Kit), and platelet-derived growth factor receptor beta were found to be 2, 12, 451, and 217 nmol/L, respectively. Ki20227 did not inhibit other kinases tested, such as fms-like tyrosine kinase-3, epidermal growth factor receptor, or c-Src (c-src proto-oncogene product). Ki20227 was also found to inhibit the M-CSF-dependent growth of M-NFS-60 cells but not the M-CSF-independent growth of A375 human melanoma cells in vitro. Furthermore, in an osteoclast-like cell formation assay using mouse bone marrow cells, Ki20227 inhibited the development of tartrate-resistant acid phosphatase-positive osteoclast-like cells in a dose-dependent manner. In in vivo studies, oral administration of Ki20227 suppressed osteoclast-like cell accumulation and bone resorption induced by metastatic tumor cells in nude rats following intracardiac injection of A375 cells. Moreover, Ki20227 decreased the number of tartrate-resistant acid phosphatase-positive osteoclast-like cells on bone surfaces in ovariectomized (ovx) rats. These findings suggest that Ki20227 inhibits osteolytic bone destruction through the suppression of M-CSF-induced osteoclast accumulation in vivo. Therefore, Ki20227 may be a useful therapeutic agent for osteolytic disease associated with bone metastasis and other bone diseases.

Bone pain is one of the major complications of bone metastases and profoundly deteriorates quality of life in cancer patients. It has been suggested that the nerve injuries by tumor invasion and the inflammatory factors secreted by cancer cells are involved in the pathophysiology of bone pain. Recent studies including ours suggest that the activation of acid-sensing receptors by tumor-induced acidosis in bone metastases also plays a part in causing bone pain. In this review, the current understanding of the mechanisms of cancer-induced bone pain is discussed.

Bone is one of the most preferential metastatic target sites for solid cancers such as breast, prostate and lung cancers. Although the precise molecular mechanisms underlying this predilection are still unclear, it appears that these cancer cells possess the capacity to modulate bone microenvironments to facilitate their arrest, survival and proliferation. In particular, production of bone-resorbing cytokines by these metastatic cancer cells increases osteoclastic bone resorption, which in turn promotes the colonization of these cancer cells in bone through releasing bone-stored growth factors into the bone marrow cavity. This crosstalk between metastatic cancer cells and bone stimulates the development and progression of bone metastases. Disruption of this vicious cycle leads us to design effective and specific interventions for osteolytic bone metastases of solid cancers.

Cyclooxygenase-2 (COX-2), the rate-limiting enzyme of prostaglandin synthesis, has been implicated in invasiveness and distant metastases of cancer. Bone is one of the most common target sites of cancer metastasis. However, the role of COX-2 in bone metastasis is unclear. We examined the surgical specimens of bone metastases from patients with various types of cancers by using immunohistochemistry and observed evident COX-2 expression in these bone metastases. In a nude mouse model of bone metastasis, the MDA-MB-231 human breast cancer cells showed no COX-2 expression at orthotopic sites, whereas these cells, when metastasized to bone, intensely expressed COX-2, suggesting that the bone microenvironment induced COX-2 expression. Consistent with this notion, inhibition of bone resorption by the bisphosphonate ibandronate reduced COX-2 expression in MDA-MB-231 cells in bone. Transforming growth factor-beta (TGFbeta), one of the most abundant growth factors stored in bone, increased COX-2 expression and prostaglandin E2 production in MDA-MB-231 cells in culture. MDA-MB-231 cells overexpressing dominant-negative TGFbeta type II receptors showed decreased bone metastases and reduced osteoclastic bone resorption with impaired COX-2 expression. The COX-2 inhibitors, NS-398 and nimesulide, significantly suppressed bone metastases with decreased osteoclast number and increased apoptosis in MDA-MB-231 cells. These results suggest that bone-derived TGFbeta up-regulates COX-2 expression in breast cancer cells, thereby increasing prostaglandin E2 production, which in turn, stimulates osteoclastic bone destruction, leading to the progression of bone metastases. Our results also suggest that COX-2 is a potential therapeutic target for bone metastases in breast cancer.

5'-deoxy-5-fluorouridine (5'-DFUR) and capecitabine are oral anti-cancer agents, which are enzymatically converted to 5-fluorouracil (5-FU) by thymidine phosphorylase in humans and uridine phosphorylase in mice. Since the activity of these phosphorylases is higher in cancerous tissue than in normal tissue, systemic administration of 5'-DFUR and capecitabine achieves high intratumoral 5-FU levels and low adverse effects on non-tumoral tissue. Accordingly, 5'-DFUR and capecitabine are widely used for the treatment of cancer patients. In the present study, we examined the effects of 5'-DFUR and capecitabine on bone metastases, one of the most common complications of breast cancer, using an animal model in which inoculation of 4T1/luc mouse breast cancer cells into the mammary fat pads of female BALB/c mice developed spontaneous metastases in distant organs including bone, lung and liver. Mice received 4T1/luc cell inoculation in the mammary fat pad at day 0 and oral 5'-DFUR (31, 62, 123 or 246 mg/kg) or capecitabine (90, 180 or 359 mg/kg) daily from day 7 to day 21. Both 5'-DFUR and capecitabine significantly inhibited orthotopic tumor formation and distant metastases to bone, lung and liver in a dose-dependent manner. Of note, the lowest dose of 5'-DFUR (31 mg/kg) and capecitabine (90 mg/kg), which failed to inhibit orthotopic tumor development and the lung and liver metastases, significantly reduced the bone metastases. In conclusion, our results suggest that oral 5'-DFUR and capecitabine are effective for the treatment of primary and secondary breast tumors. Most notably, they also suggest that these agents are preferentially beneficial for bone metastases.

Bone is one of the most preferential metastatic target sites for cancers such as breast, prostate and lung cancers. Although the precise molecular mechanisms underlying this predilection are still unclear, it appears that these cancer cells possess the capacity to modulate bone microenvironments for them to arrest, survive and proliferate. In particular, the production of bone-resorbing cytokines by these cancer cells increases osteoclastic bone resorption, which in turn facilitates the colonization of these cancer cells in bone through abundant supplies of bone-stored growth factors. This crosstalk between metastatic cancer cells and bone promotes the development and progression of bone metastases. Disruption of this vicious cycle using bisphosphonates will allow us to effectively and specifically inhibit bone metastases.

Bone, as well as lung and liver, is one of the most preferential metastatic target sites for cancers including breast, prostate, and lung cancers. Although the precise molecular mechanisms underlying this preference need to be elucidated, it appears that bone microenvironments possess unique biological features that enable circulating cancer cells to home, survive and proliferate, and destroy bone. In conjunction, cancers that develop bone metastases likely have the capacity to utilize these unique bone environments for colonization and bone destruction. This crosstalk between metastatic cancer cells and bone is critical to the development and progression of bone metastases. Disruption of this interaction will allow us to design mechanism-based effective and specific therapeutic interventions for bone metastases.

Bone sialoprotein (BSP) is a major non-collagenous protein in mineralized tissues. BSP is also implied to be involved in tumor metastasis through its unique structure. Using the human breast cancer cell line MDA-231, we established both brain-seeking and bone-seeking cell clones. The brain-seeking cells (MDA4-231BR) showed no bone metastasis in an animal model. In this experiment, MDA-231BR cells were transfected with BSP cDNA and inoculated into the hearts of nude mice. All five nude mice which received BSP-transfected MDA-231BR cells developed bone metastases, while no bone lesions were observed in the control group. Histological examination revealed invasion of tumor cells into the endosteal space and erosion of the bone margin. Some animals were crippled due to large lesions. These results suggest that BSP may impart to breast cancer cells the capacity to metastasize and thus play an important role in bone metastasis of malignant tumors.

Bone metastasis is one of the major causes of increased morbidity and eventual mortality in breast cancer patients. Therefore, intervention of bone metastases is one of the important targets in the management of breast cancer. In the present study, we examined the effects of the orally administrable chemotherapeutic agent UFT (a combination of tegafur and uracil at a molar ratio of 1:4) on bone metastases using an animal model of the 4T1/luc mouse breast cancer. The 4T1/luc cells developed spontaneous metastases to bone following orthotopic cell inoculation. Oral daily administration of UFT (20 mg/kg/day) significantly reduced the orthotopic tumor burden; however, the lower dose (15 mg/kg/day) did not. In contrast, histologic examination showed that both doses of UFT significantly suppressed bone metastases in a dose-dependent manner. Since clinical studies have demonstrated that bisphosphonates (BPs), specific inhibitors of osteoclastic bone resorption, are beneficial for breast cancer patients with bone metastases, we next examined the effects of UFT combined with the BP zoledronic acid (ZOL) on established bone metastases. The combination of UFT (20 mg/kg/day) with ZOL (250 microg/kg) caused an enhanced reduction of bone metastases compared with UFT alone. In vitro studies showed that 5-fluorouracil (5-FU), an active metabolite of UFT, and ZOL increased apoptosis in 4T1/luc cells and inhibited osteoclast-like cell formation in an additive fashion. Our results suggest that oral UFT is an effective chemotherapeutic agent for advanced breast cancer accompanying bone metastases and that the combination with BP increases its benefits for bone metastases., (Copyright 2003 Wiley-Liss, Inc.)

The proto-oncogene, c-src, has been implicated in the tumorigenesis in breast cancer. However, the relationship of c-src with distant metastasis is unclear. Moreover, the role of c-src in organ-preferential metastasis of breast cancer is unknown. Because breast cancer has a strong predilection for metastasizing to bone, we examined the role of c-src in bone metastases using an animal model in which inoculation of the MDA-231 human breast cancer cells into the left cardiac ventricle preferentially developed osteolytic bone metastases in female nude mice. A clone of the MDA-231 with the increased capacity of bone metastasis exhibited elevated c-src tyrosine kinase (TK) activity compared with parental cells. MDAsrc527 cells caused significantly increased size of the osteolytic bone metastases with increased number of osteoclasts and mitotic cancer cells compared with MDA-231EV or MDAsrcWT. In contrast, MDAsrc295 cells caused impaired metastases to bone. Of note, mice inoculated with MDAsrc295 cells via tail vein developed reduced lung metastases and prolonged survival compared with mice with MDA-231EV cells, suggesting that c-src TK is unlikely to play a specific role in bone metastases. The growth in vitro and in vivo and production of parathyroid hormone-related protein, a key cytokine in the pathogenesis of osteolytic bone metastases in breast cancer, were promoted in MDAsrc527 and diminished in MDAsrc295. These results suggest that c-src TK is associated with the capacity of breast cancer to metastasize to bone through regulating cell growth and parathyroid hormone-related protein production. Our results together with the fact that c-src is an essential molecule for bone resorption by osteoclasts, which are central players in osteolytic bone metastases, support the notion that c-src TK is a potential target molecule for designing novel therapeutic interventions, especially for bone metastases in breast cancer.

Background: Parathyroid hormone-related protein (PTH-rP) is a major cause of tumor-induced hypercalcemia (TIH) and frequently is found to be elevated in serum of patients with TIH. In the current study, the authors examined the usefulness of PTH-rP measurement at the time of first presentation in the follow-up of lung carcinoma patients with TIH., Methods: The authors retrospectively studied 23 of 1149 lung carcinoma patients who were found to have TIH at the time of first presentation for the correlation between serum PTH-rP and the development of bone metastases and survival compared with lung carcinoma patients without TIH who were matched by gender, age, Eastern Cooperative Oncology Group performance status, histological type of tumor, and stage of the disease., Results: Twenty-three lung carcinoma patients with TIH demonstrated significantly increased serum levels of PTH-rP (mean +/- standard error [SE], 84.1 +/- 16.5 pmol/L) compared with control patients without TIH (mean +/- SE, 36.2 +/- 2.0 pmol/L) at the time of first presentation, (P < 0.001). In these hypercalcemic patients, patients whose serum PTH-rP was > 150 pmol/L (n = 16) were found to have a significantly increased rate of bone metastases (71.4% vs. 12.5%; P = 0.01) and decreased survival (median survival of 1.4 months vs. 5.4 months; P < 0.015) compared with patients whose serum PTH-rP was < 150 pmol/L (n = 7)., Conclusions: The data from the current study suggest that serum PTH-rP as determined at the time of first presentation is a useful indicator of not only hypercalcemia but also bone metastasis and eventual survival in patients with lung carcinoma., (Copyright 2002 American Cancer Society.)

Bisphosphonate (BPs), specific inhibitors of osteoclastic bone resorption, are widely used therapeutic agents for bone metastases in breast cancer patients. Nevertheless, the effects of BPs on visceral metastases are controversial. Here we specifically studied the effects of the BP ibandronate on visceral metastases of breast cancer using two animal models. In the first set of experiments, we examined the effects of ibandronate on lung metastasis using 4T1 mouse mammary tumor that developed pulmonary and bone metastases following orthotopic inoculation in syngeneic female Balb/c mice. In the second set of experiments, we examined the effects of ibandronate on adrenal metastasis using a clone of the MDA-MB-231 (MDA-231) human breast cancer (MDA-231AD cells) that developed adrenal and bone metastases following intracardiac inoculation in female nude mice. These breast cancer cells were stably transfected with a firefly luciferase cDNA to facilitate quantification of the metastatic tumor burden in visceral organs. Ibandronate (4 microg/day, sc, daily) was given either after metastases were established (therapeutic administration) or at the time of tumor cell inoculation (preventative administration). In both models with each protocol, ibandronate reproducibly reduced bone metastases, establishing that BPs are effective pharmacological agents for the treatment of bone metastases in breast cancer. In the 4T1 model, neither the preventative nor therapeutic administration of ibandronate caused any effects on lung metastases. In the MDA-231 model, the preventative administration of ibandronate significantly increased adrenal metastases. However, no increase in the adrenal metastases was observed when an anti-cancer agent doxorubicin was co-administered. Therapeutic administration of ibandronate showed no effects on the adrenal metastases. Our results suggest that BPs cause no adverse effects on visceral metastases when administered in the manners that breast cancer patients usually receive.

Interleukin (IL)-18 exhibits antitumor as well as antiosteoclastogenic activities. These findings suggest that IL-18 is a potential tool for the treatment of cancers with associated osteolytic bone metastasis. We have previously shown that systemic daily administration of recombinant (r) IL-18 inhibits the development of osteolytic bone metastasis by human breast cancer cells. Here we demonstrate that systemic daily administration of rIL-18 (1 microg/mouse/d) for 21 days significantly inhibited the number and the total area of osteolytic bone metastasis by RWGT2 human lung cancer cells in nude mice. No severe adverse effects were observed. Natural killer (NK) cells did not increase in splenocytes from rIL-18-treated mice, and the in vitro NK activity of splenocytes against RWGT2 cells was only weakly enhanced in the presence of IL-18. The administration of rIL-18 made no difference in the growth of subcutaneous tumors, histologic indices (mitotic index, apoptotic index, and Ki-67-labeling index) of subcutaneous tumors or metastatic bone foci, or in the number of osteoclasts along the bone surface adjacent to tumors. Moreover, serum levels of cytokines including interferon-gamma, IL-1alpha, IL-6, tumor necrosis factor-alpha, and granulocyte/macrophage colony-stimulating factor, which regulate bone-resorbing activity of osteoclasts, were evaluated. Among them, IL-6 was remarkably downregulated in rIL-18-treated mice. These findings suggest that IL-18 inhibits osteolytic bone metastasis possibly through suppression of osteoclastic bone-resorption mediated in part by IL-6.

Breast cancer produces a variety of growth factors to promote its behavior at primary and secondary sites in autocrine/paracrine manners. However, the role of these growth factors in the colonization of cancer cells in bone, which is one of the most common metastatic sites, is poorly understood. To study this, we established an in vivo model in which the MCF-7 human breast cancer cells caused predominant osteosclerotic bone metastases 20-25 weeks after inoculation into the left cardiac ventricle in female nude mice. To make this model more time efficient, we overexpressed the oncogene Neu, which is associated with aggressive behavior in human breast cancers, in MCF-7 cells (MCF-7/Neu). MCF-7/Neu cells grew without estrogen and developed osteosclerotic bone metastases in 10-12 weeks in animals. Of note, MCF-7/Neu-bearing mice showed substantial plasma levels of human platelet-derived growth factor-BB (hPDGF-BB; 855 +/- 347 pg/ml; mean +/- SE, n = 5), indicating hPDGF-BB production by inoculated MCF-7/Neu cells. MCF-7/Neu cells in culture also produced large amounts of hPDGF-BB. Conditioned medium harvested from MCF-7/Neu cells stimulated osteoblastic bone formation in organ cultures of neonatal mouse calvariae, and a neutralizing antibody to hPDGF-BB blocked the osteoblastic bone formation. Stable transfection of the hPDGF-B AS in MCF-7/Neu cells reduced hPDGF-BB production in culture. Mice bearing these MCF-7/Neu cells with antisense showed reduced bone metastases with decreased plasma hPDGF-BB levels (54 +/- 20 and 35 +/- 21 in two different antisense and 696 +/- 312 pg/ml in empty vector; mean +/- SE; n = 5). Introduction of hPDGF-B cDNA in the MDA-MB-231 human breast cancer cells, which consistently formed osteolytic bone metastases, induced osteosclerotic lesions in the osteolytic bone metastases. In conclusion, we show that MCF-7 cells cause osteosclerotic bone metastases and that Neu enhances this capacity of MCF-7 cells. Our data suggest that MCF-7/Neu-derived hPDGF-BB plays a causative role in the development of osteosclerotic bone metastases in this model.

Breast cancer has a predilection for spreading to bone. The mechanism of preferential metastasis of breast cancer to bone is unknown. We hypothesize that breast cancer cells that develop bone metastases have the capacity to facilitate their colonization in bone. To examine this hypothesis, we established bone-seeking (MDA-231BO) and brain-seeking (MDA-231BR) clones of the human breast cancer cell line MDA-MB-231 by repeated sequential passages in nude mice and in vitro of metastatic cells obtained from bone and brain metastases, respectively. These clones were examined for distinguishing biological characteristics and compared with the MDA-231 parental cells (MDA-231P) in vivo and in vitro. Both the MDA-231BR and the MDA-231BO showed identical tumorigenicity to MDA-231P at the orthotopic site. MDA-231P that was inoculated into the heart developed metastases in bone, brain, ovary, and adrenal glands. On the other hand, MDA-231BO exclusively metastasized to bone with larger osteolytic lesions than MDA-231P. MDA-231BR exclusively disseminated to brain and failed to develop bone metastases. In culture, MDA-231BO produced greater amounts of parathyroid hormone-related protein (PTH-rP) than MDA-231BR and MDA-231P in the absence or presence of transforming growth factor beta (TGF-beta). Furthermore, the anchorage-independent growth of MDA- 231BO in soft agar was not inhibited by TGF-beta, whereas TGF-beta profoundly inhibited the growth of MDA-231P and MDA-231BR. Insulin-like growth factor I (IGF-I) markedly promoted the anchorage-independent growth of MDA-231BO, whereas marginal or no stimulation was observed in MDA-231BR or MDA-231P, respectively. Our data suggest that these phenotypic changes allow breast cancer cells to promote osteoclastic bone resorption, survive, and proliferate in bone, which consequently leads to the establishment of bone metastases.

Bisphosphonate (BP), a specific inhibitor of osteoclasts, has been widely used as a beneficial agent for the treatment of bone metastases in patients with breast cancer. It is well recognized that BP reduces osteolysis by promoting apoptosis in osteoclasts. However, recent animal and human data suggest that BPs not only reduce osteolysis associated with metastatic breast cancer, but also decrease tumor burden in bone. The mechanisms by which tumor burden is decreased following BP administration are unknown. Here we examined the effects of the BP ibandronate on MDA-231 human breast cancer cells in bone metastases in a well-characterized animal model of bone metastasis. Ibandronate, which was administered (s.c. daily; 4 microg/mouse/day) after bone metastases were established, inhibited the progression of established osteolytic bone metastases as assessed by radiographic analysis. Histological and histomorphometrical examination revealed that ibandronate reduced osteoclastic bone resorption, with increased apoptosis in osteoclasts. Furthermore, ibandronate also significantly decreased the MDA-231 tumor burden, with increased apoptosis in MDA-231 breast cancer cells in bone metastases. In contrast, ibandronate failed to inhibit MDA-231 tumor formation with no effects on apoptosis in MDA-231 breast cancer cells in the orthotopic mammary fat pads. These data suggest that the effects of ibandronate on apoptosis in MDA-231 breast cancer cells are restricted in bone in which ibandronate selectively deposits. Consistent with these in vivo results, a relatively high concentration of ibandronate (100 microM) increased caspase-3 activity and induced DNA fragmentation in MDA-231 breast cancer cells in culture. Moreover, a caspase inhibitor, z-Val-Ala-Asp-fluoromethyl ketone, blocked ibandronate-induced DNA fragmentation in MDA-231 cells, suggesting an involvement of caspase-3 in ibandronate-induced apoptosis. Our results suggest that BP suppresses bone metastases through promotion of apoptosis in metastatic cancer cells as well as in osteoclasts. However, it still remains open whether BP has direct anticancer actions in vivo.

The propensity for breast cancer cells to metastasize to bone and to induce osteolysis has long been recognized. Characteristics of both the tumor cells and the bone microenvironment contribute to this phenomenon. The presence of tumor in bone is associated with activation of osteoclasts, resulting in excessive bone resorption and subsequent osteolysis. Breast cancer cells and other tumor types influence osteoclastic bone resorption by increasing the number of osteoclasts and enhancing their resorptive activity. Parathyroid hormone-related peptide, in addition to its role in humorally mediated hypercalcemia, is secreted by metastatic breast cancer cells in bone in which it acts as a paracrine factor to stimulate osteoclasts. As bone matrix is broken down by activated osteoclasts, a rich supply of mitogenic factors is released, including insulin-like growth factors, bone morphogenetic proteins, and fibroblast growth factors. Transforming growth factor (TGF)-beta, one of the most abundant of the bone-derived factors, promotes increased production of parathyroid hormone-related peptide by tumor cells, establishing a "vicious cycle" leading to progressive tumor growth and bone destruction. Bisphosphonates interrupt this cycle by inhibiting osteoclasts, in part by inducing osteoclast apoptosis. In several animal models of breast cancer metastasis to bone, bisphosphonates decrease the number of new bone metastases and inhibit progression of existing lesions. A single 3 microg intravenous injection of zoledronic acid (Zometa; Novartis Pharmaceuticals Corp, East Hanover, NJ), a new highly potent bisphosphonate, prevented destruction of trabecular bone in an orthotopic mouse mammary tumor model. Tumor volume in bone was decreased by zoledronic acid in a dose-dependent manner in the same model, and tumor cell apoptosis was increased by zoledronic acid in bone metastases in the 4T1 murine model of mammary carcinoma metastasis. Zoledronic acid at a dose of 1.0 microg/d for 10 days also reduced bone lesion area in a nude mouse model with existing bone metastases. Although bisphosphonates, including zoledronic acid, are able to induce apoptosis in tumor cells in vitro, studies in animal models to date have generally not shown a reduction in nonosseous tumor. Therefore, bisphosphonate-associated tumor reduction in bone is most likely mediated by osteoclast inhibition or is related to high local concentrations of bisphosphonates in the bone compartment.

Background: Bone, which abundantly stores a variety of growth factors, provides a fertile soil for cancer cells to develop metastases by supplying these growth factors as a consequence of osteoclastic bone resorption. Accordingly, suppression of osteoclast activity is a primary approach to inhibit bone metastasis, and bisphosphonate (BP), a specific inhibitor of osteoclasts, has been widely used for the treatment of bone metastases in cancer patients. To obtain further insights into the therapeutic usefulness of BP, the authors studied the effects of BP on bone and visceral metastases in animal models of metastasis., Methods: The authors used two animal models of breast carcinoma metastasis that they had developed in their laboratory over the last several years. One model uses female young nude mice in which inoculation of the MDA-MB-231 or MCF-7 human breast carcinoma cells into the left cardiac ventricle selectively develops osteolytic or osteosclerotic bone metastases, respectively. Another model uses syngeneic female mice (Balb/c) in which orthotopic inoculation of the 4T1 murine mammary carcinoma cells develops metastases in bone and visceral organs including lung, liver, and kidney., Results: BP inhibited the development and progression of osteolytic bone metastases of MDA-MB-231 breast carcinoma through increased apoptosis in osteoclasts and breast carcinoma cells colonized in bone. In a preventative administration, however, BP alone increased the metastases to visceral organs with profound inhibition of bone metastases. However, combination of BP with anticancer agents such as uracil and tegafur or doxorubicin suppressed the metastases not only in bone but also visceral organs and prolonged the survival in 4T1 mammary tumor-bearing animals. Of interest, inhibition of early osteolysis by BP inhibited the subsequent development of osteosclerotic bone metastases of MCF-7 breast carcinoma., Conclusions: These results suggest that BP has beneficial effects on bone metastasis of breast carcinoma and is more effective when combined with anticancer agents. They also suggest that the animal models of bone metastasis described here allow us to design optimized regimen of BP administration for the treatment of breast carcinoma patients with bone and visceral metastases.

Bone is one of the most preferential target sites for cancer metastasis. Breast cancer has a predilection for spreading to bone, and bone metastasis is one of the major causes of increased morbidity and eventual mortality in breast cancer patients. None of the currently available therapies is effective for curing bone metastases in these patients. Elucidation of the cellular and molecular mechanism by which breast cancer selectively spreads to bone is essential for the development of mechanism-based effective and specific therapeutic interventions for this deleterious complication in breast cancer. Here, two questions are addressed to study the mechanism of breast cancer metastasis to bone: (1) What makes bone a preferential target site of metastasis? (2) What makes breast cancer able to colonize bone? (3) An animal model in which intracardiac inoculation of breast cancer cells selectively causes osteolytic bone metastases was developed. Experimental results obtained using this unique in-vivo model of bone metastasis are described and discussed.

Therapeutic effectiveness of bisphosphonates (BP) on bone metastases in patients with cancers including those of the breast and prostate has been well documented. However, there are still many important questions that remain unsolved or controversial. To obtain answers for these questions that are not readily addressed in a well-controlled manner in clinical studies, we have developed two animal models of bone metastasis (orthotopic and experimental). Using these models, we studied the effects of BP alone or in combination with anti-cancer agents on the metastasis of breast cancer to bone and visceral organs. In addition, we also determined the effects of BP on osteosclerotic metastases. We found that BP impaired the progression of bone metastases primarily through enhancing apoptosis in osteoclasts and breast cancer cells colonized in bone. In some situations, however, BP alone increased metastases in visceral organs including liver and adrenal glands. However, combination of BP with anti-cancer agents enhanced the suppression of tumour in both bone and visceral organs, leading to prolonged survival of tumour-bearing animals. Of potential importance, preventative administration of BP inhibited the development of eventual osteosclerotic bone metastases. These results suggest that BP exhibits diverse beneficial effects on osteolytic and osteoblastic bone metastasis and non-bone organ metastasis in breast cancer when administered appropriately. They also suggest that the animal models of bone metastasis described here allow us to produce clinically- relevant information that is useful for the design of optimal regimens of BP for the treatment of breast cancer patients with bone and visceral metastases., (Copyright 1999 Harcourt Publishers Ltd.)

Metastatic tumor cells can interfere directly with the function of bone cells involved in normal bone remodeling or indirectly by influencing the behavior of hematopoietic, stromal and other cells in bone marrow that interact with bone cells. Recent studies of metastatic cancer have revealed that tumor cells interact closely with vascular endothelial cells, basement membrane and bone marrow stromal cells through cell surface proteins or by releasing factors which affect the function of these cells. Bidirectional interaction between marrow cells and tumor cells can give the latter a selective advantage for growth in bone which can lead to the destruction of or to increased production of bone matrix. Understanding of the mechanisms involved in tumor metastasis and growth in bone has increased in recent years, and in this review we shall describe current knowledge of these mechanisms and of the predilection of certain types of cancers to metastasize to bone, their growth in the bone microenvironment and interactions between them and bone cells. Because metastatic breast cancer has been studied more than any other, we shall focus on it as a representative example, although the general principles apply to other types of cancer and to myeloma.

Metastasis of the oral and maxillofacial region frequently causes serious morbidity. Despite the importance of the clinical problem, little is known about the pathophysiological mechanisms of this metastatic process. Therefore, we examined whether the intracardiac injection of human breast cancer cells reproductively leads to jaw metastases developing an adequate experimental model. Human breast cancer MDA-MB-231 (MDA-231) cells (1 x 10(5)) were injected into the left heart ventricle of 4-week-old, female nude mice. Jaw metastases were examined radiographically and histologically 4 weeks after the cancer cell inoculation. At this time, the nude mice showed a marked body weight loss and cachexia. Osteolytic bone metastases were commonly observed in limbs, vertebral bone, pelvis and scapulae. In maxillofacial bones, breast cancer cells metastasized in 11 of 12 nude mice (91.7%). The lesions were radiographically determined at the mandible (11/12), maxilla (8/12) and zygomatic arch (2/12). Metastasis frequently occurred at the molar and angle regions of the mandible and at the palatal suture as well as around the root of the incisal teeth of the maxilla respectively. Histological examination revealed that numerous osteoclasts were present along the trabecular bone surfaces with aggressive bone resorption. This experimental model may be useful not only for the investigation of the mechanism of jaw metastasis formation but also for the screening of potential therapeutic agents for osteolytic bone metastasis.

Bone is one of the most preferential target organs of cancer metastases. Breast, prostate and lung cancers have a special predilection for colonization in bone. In an animal model in which inoculation of cancer cells into the left cardiac ventricle selectively develops osteolytic bone metastases but rarely forms metastases in non-bone organs, the pattern of breast cancer colonization in bone was studied radiologically and histologically. Colonization of cancer cells in bone was found to initiate and develop along with or at the terminal end of the major arteries running into bone. It should, therefore, be re-recognized that the anatomical vasculature still remains as a critical factor which influences cancer colonization in bone in addition to cellular and molecular properties of the bone microenvironment and metastatic cancer cells.

Multiple steps are involved in the metastasis of cancer cells from primary sites to distant organs. These steps should be considered in the design of pharmacologic approaches to prevent or inhibit the metastatic process. In the present study, we have compared the effects of inhibiting several steps involved in the bone metastatic process individually with inhibition of both together. The steps we chose were matrix metalloproteinase (MMP) secretion, likely involved in tumor cell invasion, and osteoclastic bone resorption, the final step in the process. We used an experimental model in which inoculation of human estrogen-independent breast cancer MDA-231 cells into the left cardiac ventricle of female nude mice causes osteolytic lesions in bone. To inhibit cancer invasiveness, the tissue inhibitor of the MMP-2 (TIMP-2), which is a natural inhibitor of MMPs, was overexpressed in MDA-231 cells. To inhibit bone resorption, a potent bisphosphonate, ibandronate (4 microg/mouse) was daily administered subcutaneously. Nude mice received either; (a) nontransfected MDA-231 cells; (b) nontransfected MDA231 cells and ibandronate; (c) TIMP-2-transfected MDA-231 cells; or (d) TIMP-2-transfected MDA-231 cells and ibandronate. In mice from group a, radiographs revealed multiple osteolytic lesions. However, in mice from group b or group c, osteolytic lesions were markedly decreased. Of particular note, in animals from group d receiving both ibandronate and TIMP-2-transfected MDA-231 cells, there were no radiologically detectable osteolytic lesions. Survival rate was increased in mice of groups c and d. There was no difference in local enlargement in the mammary fat pad between nontransfected and TIMP-2-transfected MDA-231 cells. These results suggest that inhibition of both MMPs and osteoclastic bone resorption are more efficacious treatment for prevention of osteolytic lesions than either alone, and suggest that when therapies are designed based on the uniqueness of the bone microenvironment and combined with several common steps in the metastatic process, osteolytic bone metastases can be more efficiently and selectively inhibited.