Inhibition of RANKL blocks skeletal tumor progression and improves survival in a mouse model of breast cancer bone metastasis

Developing animal models of bone metastasis in renal cell carcinoma (RCC) is challenging as immunodeficient mice are required. The aim of this study was to develop a simple immune model of RCC bone metastasis. RENCA tumor cells were injected into the right femurs of BALB/c mice. Sixty mice were grouped into each twenty-mouse group according to the tumor cell concentration, and the presence or absence and extent of bone metastasis in the total length of the femur were compared using hematoxylin and eosin staining of the excised tissues. Bone metastasis was significantly higher in the high concentration group than in the other groups (p<0.05), with 10 mice developing bone metastasis at two weeks and nine mice developing bone metastasis at three weeks. The extent of bone metastasis was significantly greater in the high concentration group than in the other groups (p<0.05). Multiple logistic regression analysis was performed to examine the factors influencing bone metastasis, and only the high concentration was a significant factor (p<0.05). We developed a normal immunity mouse model of local bone metastasis from RCC. This model could prove valuable for research into the treatment of bone metastases in RCC.

Breast cancer metastasis to bone results in significant morbidity and poor prognosis. Radium-223 dichloride is an alpha-emitting calcium mimetic that localizes to bone and provides targeted radiation therapy. A phase III clinical study on prostate cancer patients with bone metastases showed that radium-223 dichloride improved overall survival (ALSYMPCA, Parker et al. ECCO/ESMO 2011). We have previously reported that radium-223 decreases osteolysis and tumor burden in bone in a mouse model of breast cancer bone metastasis in preventive and micro-metastatic settings (Suominen et al. AACR Annual Meeting 2012), as well as, in mice with established bone metastases (Suominen et al. AACR Annual Meeting 2011). Here, we investigated the effects of radium-223 dichloride monotherapy compared to and in combination with either doxorubicin or zoledronic acid on survival in a mouse model of established breast cancer bone metastasis. Human MDA-MB-231(SA)/GFP cells were inoculated intracardially into nude mice, and 15 days later, a single dose of vehicle, radium-223 dichloride (300 kBq/kg, iv injection) and/or zoledronic acid (0.1 mg/kg, sc injection) was administered. Doxorubicin (5 mg/kg, ip injection) was administered once weekly. Radium-223 dichloride monotherapy extended time to sacrifice (P = 0.039), unlike doxorubicin or zoledronic acid monotherapy which did not improve survival as compared to the vehicle group. Radium-223 dichloride in combination with zoledronic acid (P = 0.004) or doxorubicin (P &amp;lt; 0.001) also extended time to sacrifice as compared to the vehicle but did not provide additional survival benefit as compared to the radium-223 dichloride monotherapy. Histological examination revealed that radium-223 dichloride treatment induced tumor cell necrosis in bone metastases. Therefore, the effect of radium-223 dichloride in inducing double-strand breaks in cancer cells was evaluated by immunohistochemical staining of γ-H2AX molecules. A 3-fold increase in the number of tumor cells with double-strand breaks in the radium-223 dichloride-treated as compared to the vehicle control mice was observed (P &amp;lt; 0.001). This finding supports our previous observations that radium-223 dichloride has an effect on both tumor cells and osteoclasts. In conclusion, radium-223 dichloride therapy alone or in combination with doxorubicin or zoledronic acid increases survival in breast cancer bone metastasis mouse model via dual action by targeting tumor growth and osteolysis, both important players in the destructive vicious cycle of bone metastasis. Our findings strongly support the development of radium-223 dichloride for the treatment of patients with bone metastatic breast cancer. Citation Format: Mari I. Suominen, Jukka P. Rissanen, Rami Kakonen, Katja M. Fagerlund, Esa Alhoniemi, Dominik Mumberg, Karl Ziegelbauer, Jussi M. Halleen, Sanna-Maria Kakonen, Arne Scholz. Radium-223 dichloride monotherapy and combination therapy with zoledronic acid or doxorubicin improve survival in a mouse model of breast cancer bone metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-105. doi:10.1158/1538-7445.AM2013-LB-105

Bone metastases still present an unmet need for effective treatment. One preclinical study has suggested a beneficial effect with sequential treatment of doxorubicin (DOX) and zoledronic acid (ZOL). Furthermore, bisphosphonates are used as adjuvant therapy, but controversy of their anti-tumor effects remains. We tested clinically relevant doses of DOX, ZOL and their sequential combination in a treatment setting in a widely used nude mouse model of breast cancer bone metastasis. GFP-transfected MDA-MB-231(SA) human breast cancer cells were inoculated into five-week-old female nude mice via left cardiac ventricle. Subsequent tumor growth and development of osteolytic bone lesions were quantitated by fluorescence imaging and radiography, respectively, on days 14 and 25. The mice were randomized to four groups (n=8/group) on day 14 according to body weight and the presence of osteolytic lesions. Starting on day 15, groups received either weekly dose of vehicle (control group), DOX 2.5 mg/kg i.p. weekly, ZOL 0.1 mg/kg s.c. on day 15 and vehicle weekly, or weekly dose of DOX and a single injection of ZOL 24 hrs after the first dose of DOX (combination group). Bone resorption marker tartrate-resistant acid phosphatase 5b (TRACP 5b) was determined in serum samples collected on days 1, 9, 17 and 24. Bone samples were collected on day 25, and tumor area, trabecular bone area and apoptotic index (TUNEL and apoptotic morphology) were quantitated by histomorphometry. Statistical analysis was performed using One-way ANOVA followed by Dunnett's test or Kruskal-Wallis test followed by Mann-Whitney U-test. All animals in the control and DOX groups had lesions at sacrifice, whereas 12.5% of the mice in the ZOL group and 42.9% in the combination group had lesions. Osteolytic lesion area at sacrifice was significantly lower in both groups treated with ZOL compared to the control group, with no increase from day 14. Lesion area in the DOX-treated group was not different from control. Whole body tumor burden or the tumor area in hind limbs did not differ between the groups, indicating no inhibition on tumor growth. Apoptotic index was increased in groups treated with DOX. ZOL inhibited the increase in serum TRACP 5b, although less effectively in the combination group. Taken together, clinically relevant doses of DOX, ZOL or a sequential combination treatment of these two were not able to inhibit tumor growth in the established disease in this aggressive model of breast cancer bone metastasis. However, DOX increased the apoptosis of cancer cells and ZOL potently inhibited cancer-induced osteolysis, but the sequential treatment did not have additive affects on either tumor growth or osteolytic lesion area. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5394.

Overcoming tumor tolerance and immune suppression remains a formidable obstacle to the development of immunotherapeutics that harness the immune system to eradicate breast cancer. We have developed a syngeneic mouse model to study mechanisms of tumor tolerance and immune suppression during breast cancer bone metastasis. Comparative analysis of low metastatic vs. highly bone metastatic tumor cells isolated from mice revealed several important genetic alterations related to immune control of tumor progression. Of these, a significant downregulation of cd1d1 was observed in the highly bone metastatic tumor cells. The cd1d1 gene in mice encodes the MHC class I-like molecule CD1d, which presents glycolipid antigens to a specialized subset of T cells that bridge the innate and adaptive immune response to cancer, known as invariant natural killer T (iNKT) cells. Activated iNKT cells mediate antitumor immunity in cell culture and in mouse models in vivo. Clinically, iNKT levels are significantly reduced in solid tumors, and this reduction is correlated with a poor prognosis in many types of cancers, including breast cancer. It is well known that the majority of advanced breast cancer patients present with bone metastases, and NKT cells have been shown to comprise a significant portion of the T cells found in healthy bone marrow. We hypothesize that downregulation of CD1d by breast cancer cells enables evasion of iNKT immune surveillance and inhibition of iNKT-mediated antitumor immunity, and may be an important prerequisite for the ability of breast cancer cells to colonize bone. In this study, we demonstrate in a mouse model of breast cancer bone metastasis that tumor cells expressing CD1d can induce direct iNKT-mediated cytolysis in a CD1d-dependent manner in vitro, and demonstrate increased CD1d-dependent iNKT-mediated antitumor immunity in vivo. Using transgenic knockout (KO) models, we show that the ability of CD1d-expressing tumor cells to metastasize to bone is dependent on the presence of CD1 d-restricted NKT cells. The results of this study further support research into therapeutic strategies that boost iNKT-mediated antitumor immunity for the treatment of advanced metastatic breast cancer. In addition, we propose the use of our novel mouse model of breast cancer bone metastasis as an ideal system for conducting further studies into mechanisms of immune regulation of breast cancer. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B21.

Tumor cells induce excessive osteoclastogenesis, mediating pathologic bone resorption and subsequent release of growth factors and calcium from bone matrix, resulting in a "vicious cycle" of bone breakdown and tumor proliferation. RANK ligand (RANKL) is an essential mediator of osteoclast formation, function, and survival. In metastatic prostate cancer models, RANKL inhibition directly prevents osteolysis via blockade of osteoclastogenesis and indirectly reduces progression of skeletal tumor burden by reducing local growth factor and calcium concentrations. Docetaxel, a well-established chemotherapy for metastatic hormone-refractory prostate cancer, arrests the cell cycle and induces apoptosis of tumor cells. Suppression of osteoclastogenesis through RANKL inhibition may enhance the effects of docetaxel on skeletal tumors. We evaluated the combination of the RANKL inhibitor osteoprotegerin-Fc (OPG-Fc) with docetaxel in a murine model of prostate cancer bone metastasis. Tumor progression, tumor area, and tumor proliferation and apoptosis were assessed. OPG-Fc alone reduced bone resorption (P < 0.001 versus PBS), inhibited progression of established osteolytic lesions, and reduced tumor area (P < 0.0001 versus PBS). Docetaxel alone reduced tumor burden (P < 0.0001 versus PBS) and delayed the development of osteolytic lesions. OPG-Fc in combination with docetaxel suppressed skeletal tumor burden (P = 0.0005) and increased median survival time by 16.7% (P = 0.0385) compared with docetaxel alone. RANKL inhibition may enhance docetaxel effects by increasing tumor cell apoptosis as evident by increased active caspase-3. These studies show that inhibition of RANKL provides an additive benefit to docetaxel treatment in a murine model of prostate cancer bone metastasis and supports clinical evaluation of this treatment option in patients.

Bone metastasis is a painful complication of advanced prostate cancer. Endothelin-1 is a tumor-secreted factor that plays a central role in osteoblast activation and the osteosclerotic response of prostate cancer metastatic to bone. Antagonists that block the activation of the endothelin A receptor (ETAR), located on osteoblasts, reduce osteoblastic bone lesions in animal models of bone metastasis. However, ETAR antagonists demonstrated limited efficacy in clinical trials of men with advanced prostate cancer who also received standard androgen deprivation therapy (ADT). Previous data from our group suggested that, in a mouse model, ETAR antagonists might only be efficacious when androgen signaling in the osteoblast is lowered beyond the ability of standard ADT. This notion was tested in a mouse model of prostate cancer bone metastasis. Castrated and sham-operated male athymic nude mice underwent intracardiac inoculation of the ARCaPM castration-resistant prostate cancer cell line. The mice were then treated with either the ETAR antagonist zibotentan or a vehicle control to generate four experimental groups: vehicle+sham (Veh+Sham), vehicle+castrate (Veh+Castr), zibotentan+sham (Zibo+Sham), and zibotentan+castrate (Zibo+Castr). The mice were monitored radiographically for the development of skeletal lesions. The Zibo+Castr group had significantly longer survival and a single incidental lesion. Mice in the Zibo+Sham group had the shortest survival and the largest number of skeletal lesions. Survival and skeletal lesions of the Veh+Sham and Veh+Castr groups were intermediate compared with the zibotentan-treated groups. We report a complex interaction between ETAR and androgen signaling, whereby ETAR blockade was most efficacious when combined with complete androgen deprivation.

Ewing sarcoma (ES) is a small round-cell tumor of the bones and soft tissues. ES frequently causes distant metastases, particularly in the lung and bone, which worsens patient prognosis. Cadherin-11 (Cad-11) is an adhesion molecule that is highly expressed in osteoblasts. Its expression is associated with bone metastases in prostate and breast cancer patients, and is known to occur in ES. Here we investigated the effects of Cad-11 on bone metastases of ES. Human ES cell lines RD-ES, SK-ES-1, SK-N-MC, and TC-71 cells were transduced with lentivirus containing Cad-11 shRNA or control shRNA (ES/Cad-11 and ES/Ctr). RD-ES and TC-71 were infected with a lentivirus luciferase vector. Adhesion assays were performed using these cells and recombinant Cad-11-Fc chimera or mouse osteoblast cell line MC3T3-E1. Cell motility was investigated via wound-healing assay. Intracardiac injection of RD-ES/Cad-11 and RD-ES/Ctr was used to create a mouse model of experimental bone metastasis. The association between Cad-11 expression and bone metastases and clinical prognosis in ES patients was analyzed by immunohistochemistry. We found knockdown of Cad-11 in ES cells resulted in reduced attachment ability and cell motility. In a mouse model of metastasis, RD-ES/Cad-11 cells caused fewer metastases than RD-ES/Ctr cells. The expression of Cad-11 in ES patients was significantly related to bone metastases (P < 0.05, logistic regression) and poorer overall survival (P < 0.05, log-rank test). These findings may explain that Cad-11 in ES cells may be essential for cell adhesion and motility, and is a promising molecular target for patients with ES.

Introduction: The process of bone metastasis involves both osteoblastic and resorptive components and is a common feature of both prostate and breast cancers. The ability to visualize and quantify the early stages of bone involvement in mouse models of bone metastasis would provide a platform for development of new agents targeted at inhibition or treatment of bone metastases. Two approaches that stand to enable this are 18F-NaF PET and optical imaging using biphosphonate fluorescent probes such as Osteosense, through targeting of hydroxyapatite (HA), a biomarker for osteoblastic activity. Additionally, bioluminescence imaging using luciferase expressing tumor lines and micro-CT imaging of the skeleton can enable anatomic characterization of tumor burden and development of bone lesion in bone metastasis models. Methods: MDA-MB-231-luc-D3H2LN human mammary adenocarcinoma cells (105 cells in 100µl) were injected into the left ventricle of female nu/nu mice on Day 0 approximately 6-7min after IP injection of luciferin (150mg/kg). Bioluminescence scans were used to determine successful injection by distribution of light throughout the body. On Day 14, all tumor pool mice were imaged using bioluminescence and enrolled on study based on incidence of luciferase signals at bone sites. After staging, serial bioluminescence imaging was used to localize tumor signals and determine incidence and growth of bone metastases and micro-CT was used to image the extent of bone lesions associated with the metastases. Additionally, whole body PET scans were used at multiple time points after 90min uptake of 18F-NaF. Images were reconstructed using a 2D OSEM method and localized bone sites segmented and analyzed for standardized uptake value (SUV). Fluorescence imaging was also used 24h after administration of Osteosense 750 to characterize localization of the tracer at the bone sites of interest. After background correction, the bone sites of interest were segmented and analyzed for average efficiency. The 18F-NaF and Osteosense endpoints were correlated with bioluminescence determined tumor burden and location. Results and Discussion: Both 18F-NaF PET imaging and fluorescent imaging using Osteosense highlighted localized bone signals that could be correlated with bioluminescent signals and micro-CT visualized bone lesions from approximately Day 17. Both the PET and fluorescence imaging approaches showed early indication of bone involvement, presumably through HA, indicating osteoblastic activity. This multi-endpoint approach enabled non-invasive determination of both soft tumor and bone components of metastatic bone disease. An approach like this may enable more quantitative characterization of the early stages of bone metastasis in mouse models, and facilitate development of bone targeted treatments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4321.

Background: Bone metastasis in human prostate cancer remains a major clinical problem since no effective therapy exists. The RANKL/RANK pathway plays a predominant role in the interaction between metastasized prostate cancer cells and osteoclasts that increases the bone turnover. The current therapies, including targeting RANKL with denosumab, address the growth of prostate tumor cells that have already colonized the bone, but are largely ineffective in prolonging the survival of human prostate cancer patients with bone metastasis. Further, a major impediment to prostate cancer bone metastasis research is the lack of an animal model that spontaneously recapitulates human prostate cancer bone metastasis in the context of an intact immune system. Results: To overcome this major limitation, we have developed a novel syngeneic mouse model to study prostate cancer bone metastasis. Both the CXCL12/CXCR4 and RANKL/RANK pathways have been reported to be overexpressed / dysregulated in human prostate cancer bone metastatic samples. Data generated utilizing our immune-intact mouse model shows that the CXCL12/CXCR4 and RANKL/RANK pathways co-operate with each other to drive prostate cancer bone metastasis. Studies have shown that targeting the CXCL12/CXCR4 and RANKL/RANK pathways individually affects the immune system, thereby making our syngeneic mouse model an indispensable tool for studying the critical co-operation between these 2 pathways in the manifestation of human prostate cancer bone metastasis. Extending our earlier findings that RANKL drives PCa metastases in immune-deficient mice (Chu et al., 2014) to immune-intact C57/Bl6 mice, we found that MPC3 mouse PCa cells with RANKL overexpression (MPC3-Luc-GFP-RANKL) develop 70-80% limb and jaw within 4 weeks of intra-cardiac injection in these syngeneic mice. Control MPC3 cells had no bone metastasis. Bone lesions visualized by luciferase imaging and X-ray were confirmed by micro CT and immunohistochemistry. RANKL signaling drove bone and visceral metastases via the downstream CXCL12/CXCR4 signaling axis. MPC3-Luc-GFP-RANKL cells showed increased CXCR4 protein levels by immunohistochemistry. Metastatic bone marrow flush showed dramatically increased levels of CXCL12 mRNA compared with control mice (MPC3-Luc-GFP-EV). Conclusion: In sum, 1) circulating PCa cells induce a marked CXCL12 elevation after colonizing bone, triggering chemotaxis and recruiting CXCR4-positive PCa cells to migrate to bone; and 2) osteomimetic PCa cells with increased RANKL expression interact with osteoclasts to enhance bone resorption and turnover, releasing additional growth factors and chemokines for PCa cell growth and survival in bone. Citation Format: Srinivas Nandana, Murali Gururajan, Manisha Tripathi, Chia-Yi Chu, Haiyen Zhau, Stephen Shiao, Leland Chung. A novel syngeneic mouse model of prostate cancer bone metastasis: Mechanisms of chemotaxis and bone colonization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4523.

Raman spectroscopy of bone has been used to characterize chemical changes occurring in diseases such as osteoporosis, osteoarthritis and osteomyelitis. Metastasis of cancer into bone causes changes to bone quality that are similar to those observed in osteoporosis, such as decreased bone strength, but with an accelerated timeframe. In particular, osteolytic (bone degrading) lesions in bone metastasis have a marked effect on patient quality of life because of increased risk of fractures, pain, and hypercalcemia. We use Raman spectroscopy to examine bone from two different mouse models of osteolytic bone metastasis. Raman spectroscopy measures physicochemical information which cannot be obtained through standard biochemical and histological measurements. This study was reviewed and approved by the University of Michigan University Committee on the Care and Use of Animals. Two mouse models of prostate cancer bone metastasis, RM1 (n=3) and PC3-luc (n=4) were examined. Tibiae were injected with RM1 or PC3-luc cancer cells, while the contralateral tibiae received a placebo injection for use as controls. After 2 weeks of incubation, the mice were sacrificed and the tibiae were examined by Raman microspectroscopy (&lambda;=785 nm). Spectroscopic markers corresponding to mineral stoichiometry, bone mineralization, and mineral crystallinity were compared in spectra from the cancerous and control tibiae. X-ray imaging of the tibia confirmed extensive osteolysis in the RM1 mice, with tumor invasion into adjoining soft tissue and moderate osteolysis in the PC3-luc mice. Raman spectroscopic markers indicate that osteolytic lesions are less mineralized than normal bone tissue, with an altered mineral stoichiometry and crystallinity.

&lt;div&gt;Abstract&lt;p&gt;TGF-β regulates several steps in cancer metastasis, including the establishment of bone metastatic lesions. TGF-β is released from bone during osteoclastic bone resorption and it stimulates breast cancer cells to produce osteolytic factors such as interleukin 11 (IL-11). We conducted a cell-based siRNA screen and identified heparan sulfate 6-&lt;i&gt;O&lt;/i&gt;-sulfotransferase 2 (HS6ST2) as a critical gene for TGF-β–induced IL-11 production in highly bone metastatic MDA-MB-231(SA) breast cancer cells. HS6ST2 attaches sulfate groups to glucosamine residues in heparan sulfate glycosaminoglycans. We subsequently showed how heparin and a high-molecular-weight &lt;i&gt;Escherichia coli&lt;/i&gt; K5-derived heparin-like polysaccharide (K5-NSOS) inhibited TGF-β–induced IL-11 production in MDA-MB-231(SA) cells. In addition, K5-NSOS inhibited bone resorption activity of human osteoclasts &lt;i&gt;in vitro&lt;/i&gt;. We evaluated the therapeutic potential of K5-NSOS and fragmin in a mouse model of breast cancer bone metastasis. MDA-MB-231(SA) cells were inoculated into the left cardiac ventricle of athymic nude mice which were treated with fragmin, K5-NSOS, or vehicle once a day for four weeks. Both heparin-like glycosaminoglycans inhibited weight reduction, decreased osteolytic lesion area, and reduced tumor burden in bone. In conclusion, our data imply novel mechanisms involved in TGF-β induction and support the critical role of heparan sulfate glycosaminoglycans in cancer metastasis as well as indicate that K5-NSOS is a potential antimetastatic and antiresorptive agent for cancer therapy. This study illustrates the potential to translate &lt;i&gt;in vitro&lt;/i&gt; siRNA screening results toward &lt;i&gt;in vivo&lt;/i&gt; therapeutic concepts. &lt;i&gt;Mol Cancer Res; 10(5); 597–604. ©2012 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

TGF-β regulates several steps in cancer metastasis, including the establishment of bone metastatic lesions. TGF-β is released from bone during osteoclastic bone resorption and it stimulates breast cancer cells to produce osteolytic factors such as interleukin 11 (IL-11). We conducted a cell-based siRNA screen and identified heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) as a critical gene for TGF-β-induced IL-11 production in highly bone metastatic MDA-MB-231(SA) breast cancer cells. HS6ST2 attaches sulfate groups to glucosamine residues in heparan sulfate glycosaminoglycans. We subsequently showed how heparin and a high-molecular-weight Escherichia coli K5-derived heparin-like polysaccharide (K5-NSOS) inhibited TGF-β-induced IL-11 production in MDA-MB-231(SA) cells. In addition, K5-NSOS inhibited bone resorption activity of human osteoclasts in vitro. We evaluated the therapeutic potential of K5-NSOS and fragmin in a mouse model of breast cancer bone metastasis. MDA-MB-231(SA) cells were inoculated into the left cardiac ventricle of athymic nude mice which were treated with fragmin, K5-NSOS, or vehicle once a day for four weeks. Both heparin-like glycosaminoglycans inhibited weight reduction, decreased osteolytic lesion area, and reduced tumor burden in bone. In conclusion, our data imply novel mechanisms involved in TGF-β induction and support the critical role of heparan sulfate glycosaminoglycans in cancer metastasis as well as indicate that K5-NSOS is a potential antimetastatic and antiresorptive agent for cancer therapy. This study illustrates the potential to translate in vitro siRNA screening results toward in vivo therapeutic concepts.

Bone metastases are associated with increased morbidity and poor prognosis in breast cancer patients. Radium-223 dichloride is a calcium mimetic that localizes to bone, providing targeted therapy for skeletal metastasis. We investigated the mode of action of radium-223 dichloride using breast cancer cell, osteoclast, and osteoblast cultures as well as a mouse model of breast cancer bone metastasis. A single dose of radium-223 dichloride was used in three different settings mimicking the prevention or treatment of bone metastasis. Disease progression was monitored using fluorescence and radiographic imaging and histological analyses. The effect of radium-223 dichloride alone and in combination with doxorubicin or zoledronic acid on survival of mice was analyzed by Kaplan-Meier methods. All statistical tests used were two-sided. Radium-223 dichloride incorporated into bone matrix and inhibited proliferation of breast cancer cells and differentiation of osteoblasts and osteoclasts (all P values < .001) in vitro. In an established bone metastasis setting, radium-223 dichloride prevented tumor-induced cachexia (0/14 vs 7/14 control mice) and decreased osteolysis by 56% and tumor growth by 43% (all P values < .05). Radium-223 dichloride induced double-strand DNA breaks in cancer cells in vivo. Finally, radium-223 dichloride extended survival as a monotherapy (29.2 days, 95% confidence interval [CI] = 26.6 to 31.8 days, P = .039) and in combination with zoledronic acid (31.4 days, 95% CI = 28.8 to 34.0 days, P = .004) or doxorubicin (31.5 days, 95% CI = 29.5 to 33.5 days, P < .001) compared to the vehicle group (24.9 days, 95% CI = 23.4 to 26.4 days). Similar but even more pronounced effects were observed when radium-223 dichloride was administered in a preventive or micrometastatic setting. Our findings strongly support the development of radium-223 dichloride for the treatment of breast cancer patients with or at high risk of developing bone metastases.

Transforming growth factor β (TGF-β) is a key regulator of several steps in cancer metastasis, including the establishment of bone metastatic lesions. TGF-β is released from bone during osteoclastic bone resorption and it stimulates breast cancer cells to produce osteolytic factors such as interleukin 11 (IL-11). We conducted a cell-based siRNA screen and identified heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) as a critical gene for TGF-β-induced IL-11 production in highly bone metastatic MDA-MB-231(SA) breast cancer cells. HS6ST2 attaches sulfate groups to glucosamine residues in heparin sulfate which is a member of the glycosaminoglycan family of carbohydrates. Our subsequent studies with highly sulfated heparin-like glycosaminoglycans (HLGAGs) with low anti-coagulant activity indicated that a high-molecular-weight E. coli K5-derived heparin-like polysaccharide (K5-NSOS) inhibited TGF-β-induced IL-11 production in MDA-MB-231(SA) cells. We then evaluated the therapeutic potential of K5-NSOS and a low-molecular-weight synthetic heparin (fragmin) in a mouse model of breast cancer bone metastasis. MDA-MB-231(SA) cells were inoculated into the left cardiac ventricle of athymic nude mice which were then administered with K5-NSOS, fragmin, or vehicle once daily for 4 weeks. Both HLGAGs increased body weight, decreased osteolytic lesion area, and reduced tumor burden in bone. Our in vitro studies showed that both K5-NSOS and fragmin inhibited adhesion of breast cancer cells to ICAM-1, and K5-NSOS also inhibited adhesion to E- and P-selectin. Furthermore, K5-NSOS inhibited bone resorption activity of human osteoclasts in vitro. Our data support and further clarify the critical role of heparan sulfate glycosaminoglycans in breast cancer metastasis and indicate that K5-NSOS is a potential antimetastatic and antiresorptive agent with low anticoagulant activity that could be further optimized as an anti-tumor agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 845. doi:10.1158/1538-7445.AM2011-845

&lt;div&gt;Abstract&lt;p&gt;TGF-β regulates several steps in cancer metastasis, including the establishment of bone metastatic lesions. TGF-β is released from bone during osteoclastic bone resorption and it stimulates breast cancer cells to produce osteolytic factors such as interleukin 11 (IL-11). We conducted a cell-based siRNA screen and identified heparan sulfate 6-&lt;i&gt;O&lt;/i&gt;-sulfotransferase 2 (HS6ST2) as a critical gene for TGF-β–induced IL-11 production in highly bone metastatic MDA-MB-231(SA) breast cancer cells. HS6ST2 attaches sulfate groups to glucosamine residues in heparan sulfate glycosaminoglycans. We subsequently showed how heparin and a high-molecular-weight &lt;i&gt;Escherichia coli&lt;/i&gt; K5-derived heparin-like polysaccharide (K5-NSOS) inhibited TGF-β–induced IL-11 production in MDA-MB-231(SA) cells. In addition, K5-NSOS inhibited bone resorption activity of human osteoclasts &lt;i&gt;in vitro&lt;/i&gt;. We evaluated the therapeutic potential of K5-NSOS and fragmin in a mouse model of breast cancer bone metastasis. MDA-MB-231(SA) cells were inoculated into the left cardiac ventricle of athymic nude mice which were treated with fragmin, K5-NSOS, or vehicle once a day for four weeks. Both heparin-like glycosaminoglycans inhibited weight reduction, decreased osteolytic lesion area, and reduced tumor burden in bone. In conclusion, our data imply novel mechanisms involved in TGF-β induction and support the critical role of heparan sulfate glycosaminoglycans in cancer metastasis as well as indicate that K5-NSOS is a potential antimetastatic and antiresorptive agent for cancer therapy. This study illustrates the potential to translate &lt;i&gt;in vitro&lt;/i&gt; siRNA screening results toward &lt;i&gt;in vivo&lt;/i&gt; therapeutic concepts. &lt;i&gt;Mol Cancer Res; 10(5); 597–604. ©2012 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;