Abstract 845: Heparin-like polysaccharides reduce osteolytic bone destruction and tumor growth in a mouse model of breast cancer bone metastasis

<div>Abstract<p>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-<i>O</i>-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 <i>Escherichia coli</i> 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 <i>in vitro</i>. 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 <i>in vitro</i> siRNA screening results toward <i>in vivo</i> therapeutic concepts. <i>Mol Cancer Res; 10(5); 597–604. ©2012 AACR</i>.</p></div>

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.

<div>Abstract<p>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-<i>O</i>-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 <i>Escherichia coli</i> 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 <i>in vitro</i>. 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 <i>in vitro</i> siRNA screening results toward <i>in vivo</i> therapeutic concepts. <i>Mol Cancer Res; 10(5); 597–604. ©2012 AACR</i>.</p></div>

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 < 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 < 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.

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.

Bone metastases cause severe skeletal morbidity including fractures and hypercalcemia. Tumor cells in bone induce activation of osteoclasts, which mediate bone resorption and release of growth factors from bone matrix, resulting in a "vicious cycle" of bone breakdown and tumor proliferation. Receptor activator of NF-kappaB ligand (RANKL) is an essential mediator of osteoclast formation, function, and survival, and is blocked by a soluble decoy receptor, osteoprotegerin (OPG). In human malignancies that metastasize to bone, dysregulation of the RANK/RANKL/OPG pathway can increase the RANKL:OPG ratio, a condition which favors excessive osteolysis. In a mouse model of bone metastasis, RANKL protein levels in MDA-MB-231 (MDA-231) tumor-bearing bones were significantly higher than tumor-free bones. The resulting tumor-induced osteoclastogenesis and osteolysis was dose-dependently inhibited by recombinant OPG-Fc treatment, supporting the essential role for RANKL in this process. Using bioluminescence imaging in a mouse model of metastasis, we monitored the anti-tumor efficacy of RANKL inhibition on MDA-231 human breast cancer cells in a temporal manner. Treatment with OPG-Fc in vivo inhibited growth of MDA-231 tumor cells in bony sites when given both as a preventative (dosed day 0) and as a therapeutic agent for established bone metastases (dosed day 7). One mechanism by which RANKL inhibition reduced tumor burden appears to be indirect through inhibition of the "vicious cycle" and involved an increase in tumor cell apoptosis, as measured by active caspase-3. Here, we demonstrate for the first time that OPG-Fc treatment of mice with established bone metastases resulted in an overall improvement in survival.

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.

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.

We have previously developed a genetically-modified bacterial strain of Salmonella typhimurium (A1-R), which expresses GFP. In this study, we measured the efficacy of A1-R on breast cancer bone metastasis. We established an early-stage bone-metastasis model in nude mice by cardiac injection and a late-stage model by injection into the intramedullary cavity of the tibia using MDA-MB-435 human breast cancer cells (Human breast cancer cell lines co-express neuronal, epithelial, and melanocytic differentiation markers in vitro and in vivo. PLoS One 5, e9712, 2010). Both mouse models of bone metastasis were treated with A1-R. Fluorescence imaging of the mice was performed to visualize the metastatic bone lesions. In a first set of experiments, we showed that A1-R invaded and replicated intracellularly in MDA-MB-435-GFP cells in vitro. A1-R dose-dependently inhibited proliferation of MDA-MB-435-GFP cells. In the early-stage bone-metastasis model, A1-R significantly improved metastasis-free survival. In the advanced-stage bone-metastasis model, A1-R significantly inhibited the growth of the metastatic lesions. These data indicated that A1-R is useful to prevent and inhibit the growth of metastatic breast cancer bone tumors. Citation Format: Shinji Miwa, Ming Zhao, Shuya Yano, Yong Zhang, Fuminari Uehara, Yasunori Matsumoto, Yukihiko Hiroshima, Mako Yamamoto, Hiroaki Kimura, Katsuhiro Hayashi, Hiroyuki Tsuchiya, Robert M. Hoffman. The efficacy of tumor-targeting Salmonella typhimurium A1-R on bone metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 710. doi:10.1158/1538-7445.AM2014-710

In breast cancer patients, disseminated tumor cells can maintain in dormancy at secondary organs for a long period before relapse into life-threatening metastases. However, the molecular understanding of this process is still very limited. Here, we show that Vascular Cell Adhesion Molecule-1 (VCAM1) is essential for the conversion from dormancy to macrometastasis in a xenograft mouse model of breast cancer bone metastasis. A weakly bone-metastatic subline (SCP6) of the human breast cancer cell MDA-MB-231 was found to give rise to strongly metastatic derivatives after six-month in vivo dormancy in nude mice. Whereas no additional chromosomal alterations were detected by array-CGH, the derivatives significantly upregulated a handful of genes based on microarray analysis. Five candidate genes were evaluated by silencing their expression using short-hairpin RNA in the highly metastatic derivatives and tested by in vivo bone metastasis assay. Only the loss of expression of VCAM1 dramatically reduced the metastasis ability. Ectopic overexpression of VCAM1 in dormancy-prone sublines allowed the outgrowth of bone metastasis. We successfully blocked the initiation and the progression of bone metastasis using purified monoclonal antibodies against VCAM1 or its receptor VLA4 (integrin α4β1). Biochemical analysis showed activated NF-κB pathway is essential for the upregulation of VCAM1. Bone metastases formed by VCAM1-overexpressed tumor cells harbored numerous TRAP-positive osteoclasts and their number was dramatically attenuated with VCAM1 or VLA4 antibodies. Expression analysis using cell lines and fresh murine bone marrow showed progenitor and mature osteoclasts express α4β1. Functional assays found VCAM1 promoted osteoclast differentiation by directly tethering the α4β1-positive osteoclast progenitors. Mice with VCAM1-positive bone metastases showed enriched CD11b-positive α4β1-positive monocytic progenitor cells of osteoclasts in the bone marrow compared with mice free of bone metastases. Finally, tissue microarray analysis showed VCAM1 is upregulated in breast tumor epithelial cells in a subgroup of patients and is associated with early relapse. Overall, our study (1) established a novel mouse model for bone metastasis dormancy of breast cancer; (2) demonstrated VCAM1 as the key mediator for the conversion from dormancy to overt metastasis through engaging α4β1-positive osteoclast progenitors; (3) developed monocloncal antibodies against VCAM1 or VLA4 as promising therapeutic agents for preventing and treating bone metastasis of breast cancer. 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 5763.

The Role of Post-Menopausal Osteoporosis in Bone Metastatic Breast Cancer and the Effect of Monocyte Chemoattractant Protein-1

Macrophages of the tumor microenvironment can facilitate cancer progression and are a possible target for therapy. We have developed PLX3397, an orally-active, selective small molecule inhibitor of the colony stimulating factor-1 receptor (CSF-1R) kinase. CSF-1R is known to control a key signaling pathway for the recruitment of macrophages to tumors, and also for differentiation of osteoclasts mediating the destructive processes of bone metastasis. We show efficacy of PLX3397 in three different animal cancer models. PLX3397, which is currently in Phase I clinical trial, also inhibits the related kinase Kit, although we believe the efficacy observed here primarily reflects CSF-1R inhibition. SK-N-SH human neuroblastoma cells grown as xenografts in female nude mice were inhibited 60% in their growth compared to controls after 18 days oral dosing of PLX3397. This contrasts with a very weak effect on growth of SK-N-SH cells in culture, with an IC50 of 10 μM. A similar lack of growth inhibitory effects of PLX3397 was obtained using several cancer cells and when tested in cell toxicity assays. In a separate study, PLX3397 had little or no effect on the growth of MDA-MB-231 human tumor cells grown as xenografts. These results suggest that the observed inhibition of SK-N-SH cells grown in vivo are mediated by targeting cells of the microenvironment, and further, that this neuroblastoma may be more dependant on the microenvironment than other tumor cells. The MMTV-PyMT transgenic mouse breast cancer model has been used previously to identify mechanisms through which tumor-associated macrophages can enhance metastasis. Using this model, PLX3397 caused a 90% reduction in circulating tumor cells (CTCs), quantified by FACS analysis with a pan-cytokeratin antibody. This decrease was observable after two oral doses given at 18 and 4 hours before a terminal blood sample was taken. A similar decrease in CTCs was observed using QPCR in syngeneic mice orthotopically implanted with a tumor cell line derived from the transgenic mice. PLX3397 also worked at low concentrations in vitro to reduce by 55% the ability of macrophages (purified from tumors) to induce invasive acini in breast cancer cells grown in culture as spheroids. MDA-MB-468 human breast tumor cells injected into the tibia of female SCID mice caused trabecular bone loss as measured by micro-CT, and this bone lysis was associated with a 4-fold elevation in osteoclast number. Oral dosing of PLX3397 prevented both the rise in osteoclasts and the loss of bone, suggesting this compound is able to inhibit the intended CSF-1R target in vivo, and predicting it may offer benefit to cancer patients suffering with bone metastasis. 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 3850.

TGF-β manifests multifunctional and dichotomous roles in different stages of cancer progression. In premalignant cells, TGF-β is primarily a tumor suppressor that inhibits cell proliferation or induces apoptosis. In the later stages of cancer progression, however, TGF-β functions as a metastasis promoter by inducing epithelial-mesenchymal transition (EMT), leading to increased invasion of cancer cells, and also by inducing genes that facilitate metastatic colonization of secondary organ sites. Although the opposing functions of TGF-β in early- versus late-stage cancer have been known for decades, how and when TGF-β switches its functional roles are long-standing questions with no clear answer. It has been postulated that the dichotomous functions of TGF-β are dictated by different partners of its downstream Smads. However, it is unclear how Smad partners are changed in different stages of cancer. We have investigated how Smad partners are changed in different stages of breast cancer development in 2D and 3D cell cultures by both hypothesis-driven and unbiased approaches, in mouse models of bone metastasis, by bioinformatics analysis of breast cancer TCGA data, and by immunohistochemistry analysis of different stages of human breast diseases/cancers. Together, our data demonstrated that 14-3-3ζ overexpression induces contextual changes of Smad partners. Specifically, 14-3-3ζ 1) switches off TGF-β's cytostatic tumor suppressor function during the early stage (ADH) of breast cancer development by cytoplasmic sequestration of YAP1, leading to reduced 14-3-3σ transcription, which results in destabilization of p53, a Smad determinant for p21 transactivation in pre-malignant cells, and consequently decreased p21 expression; 2) switches on TGF-β's metastasis promoter function during breast cancer progression (DCIS/IDC) by blocking Gli2 binding with its E3 ligase β-TrCP, thus stabilizing Gli2, a Smad determinant for PTHrP transactivation in cancer cells, leading to bone metastasis (Cancer Cell, In Press, 2015). The critical role of TGF-β in cancer, especially in the process of metastasis, has spurred the development of TGF-β-targeting agents as cancer therapeutics. Disappointingly, many of the current TGF-β-targeting drugs showed limited clinical efficacy. Considering the opposing functions of TGF-β in cancer development, general inhibition of the TGF-β pathway may have deleterious consequence. Based on our new findings, the 14-3-3ζ-driven contextual changes of Smad partners from p53 to Gli2 may serve as a) biomarkers and b) therapeutic targets of TGF-β-mediated cancer progression and metastasis. Citation Format: Jia Xu, Sunil Acharya, Ozgur Sahin, Lin Zhang, Frank J. Lowery, Aysegul A. Sahin, Xiang H.-f. Zhang, Mien-Chie Hung, Dihua Yu. 14-3-3ζ turns TGF-β's function from tumor suppressor to metastasis promoter in breast cancer by contextual changes of Smad partners from p53 to Gli2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-202. doi:10.1158/1538-7445.AM2015-LB-202

Bone is the most frequent site for breast cancer metastasis. Metastatic cancer cells secrete parathyroid-hormone related peptide (PTHrP) that stimulates bone turnover, releasing growth factors and creating a cavity for tumor growth. Current treatments include bisphosphonates and the RANK Ligand inhibitors that have limited efficacy. PTHrP antagonists have failed clinically due to rapid drug turnover and kidney side effects causing hypocalcemia. We synthesized two novel PTHrP antagonists that are fused to an inert bacterial collagen binding domain (CBD) of colG collagenase from Clostridium histolyticum. The collagen binding activity directs PTH analogs to the bone matrix where it blocks PTHrP action. PTH(7-33)-CBD is an amino truncated PTHrP antagonist fused to the CBD that we previously showed reduced metastatic tumor burden in bone and prevented bone destruction. We now compare PTH(7-33)-CBD to [W2]PTH(1-33)-CBD, an inverse agonist at the PTH/PTHrP receptor. In vitro neither drug induced PTH receptor activation in SaOS-2 osteosarcoma cells as measured by cAMP accumulation. Both compounds partially antagonized PTH(1-34) agonist-induced cAMP accumulation. Importantly, [W2]PTH(1-33)-CBD reduced agonist-stimulated cAMP accumulation by >95% (vs. 71% for PTH(7-33)-CBD) exceeding that reported previously for any PTH antagonist. Both compounds induced apoptosis of MDA-MB-231 breast cancer cells in vitro. In vivo testing was performed with an established mouse model of breast cancer bone metastasis using a bone-trophic variant of MDA-MB-231 breast cancer cells expressing luciferase (MDA-MB-231-BM/luc+). 24 hours after drug administration (1000 µg/kg administered IP), MDA-MB-231-BM/luc+ cells were injected intratibially into nude mice. PTH(7-33)-CBD significantly reduced tumor burden in bone (P<0.05) compared to both vehicle control (VC) (weeks 4-8), and to control PTH(7-34) antagonist that lacks the CBD (weeks 6-7). [W2]PTH(1-33)-CBD showed a trend towards decrease in tumor burden but did not reach statistical significance. Both PTH(7-33)-CBD and [W2]PTH(1-33)-CBD significantly reduced osteolytic lesions (P<0.05) compared to VC and PTH(7-34) weeks 3-8 and weeks 6-8, respectively. Drug treatments did not significantly affect animal weight nor alter serum calcium, alkaline phosphatase or N-terminal propeptide of Type 1 procollagen (PN1P) bone formation marker. Tartrate resistant acid phosphatase (TRAP), a marker of bone removal, was significantly reduced by treatments. In summary, a new PTHrP inverse agonist, [W2]PTH(1-33)-CBD, partially suppressed basal signaling of the PTH/PTHrP receptor and demonstrated a higher degree of antagonist activity in-vitro than any PTH antagonist published to date. It is expected that the superior antagonist activity of [W2]PTH(1-33)-CBD will provide significant tumor protection against breast cancer metastasis to bone.