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Metabolic Syndrome (MetS) has detrimental effects on the bladder, including detrusor underactivity. The progression and mechanism of disease are poorly understood. A swine model for diabetic bladder dysfunction (DBD) was established because of the pig's human-sized bladder and its ability to develop MetS by dietary modification alone. The hypothesis of this study is that this swine model will demonstrate oxidative stress associated with MetS, which contributes to both bladder fibrosis and detrusor underactivity (DU). Ossabaw pigs underwent dietary modification consisting of a hypercaloric, atherogenic diet for 10 mo to induce MetS, and were compared with a group of control (lean) pigs. Urodynamic studies were performed in both groups to confirm DU. Thiobarbituric acid reactive substances (TBARS) detected in the urine were used to measure oxidative stress activity in the urinary tract, and urinary IL17a was used to detect profibrotic activity. MetS was confirmed by assessing body weight, blood pressure, glucose tolerance, total cholesterol, and triglycerides. The MetS group exhibited an increase in the relative levels of urinary TBARS and IL17a. Bladder pressures at capacity were lower in the MetS group, suggesting DU. Histologic analysis of a cohort of control (lean) and MetS pigs revealed that as compared with the control pigs, the MetS pigs had significantly more collagen in the muscularis layer, but not in the submucosa or mucosa layer. In conclusion, the Ossabaw pig model for diet-induced MetS is associated with oxidative stress and profibrotic activity in the bladder, which results in DU. This has previously been shown in mice and rats, but never in pigs. This novel model will better represent human MetS and DBD because the mechanism and size of the pig bladder more closely resemble that of a human, resulting in a more valid model and facilitating further study into the signaling mechanisms responsible for this impairment.

Complications associated with advanced cancer are a major clinical challenge and, if associated with bone metastases, worsen the prognosis and compromise the survival of the patients. Breast and prostate cancer cells exhibit a high propensity to metastasize to bone. The bone microenvironment is unique, providing fertile soil for cancer cell propagation, while mineralized bone matrices store potent growth factors and cytokines. Biologically active transforming growth factor β (TGF-β), one of the most abundant growth factors, is released following tumor-induced osteoclastic bone resorption. TGF-β promotes tumor cell secretion of factors that accelerate bone loss and fuel tumor cells to colonize. Thus, TGF-β is critical for driving the feed-forward vicious cycle of tumor growth in bone. Further, TGF-β promotes epithelial-mesenchymal transition (EMT), increasing cell invasiveness, angiogenesis, and metastatic progression. Emerging evidence shows TGF-β suppresses immune responses, enabling opportunistic cancer cells to escape immune checkpoints and promote bone metastases. Blocking TGF-β signaling pathways could disrupt the vicious cycle, revert EMT, and enhance immune response. However, TGF-β’s dual role as both tumor suppressor and enhancer presents a significant challenge in developing therapeutics that target TGF-β signaling. This review presents TGF-β’s role in cancer progression and bone metastases, while highlighting current perspectives on the therapeutic potential of targeting TGF-β pathways.

// Patricia Juarez 1, 2 , Pierrick G.J. Fournier 1, 2 , Khalid S. Mohammad 1 , Ryan C. McKenna 3 , Holly W. Davis 3 , Xiang H. Peng 1 , Maria Niewolna 1 , Alain Mauviel 4, 5, 6, 7 John M. Chirgwin 1 and Theresa A. Guise 1 1 Division of Endocrinology, Department of Medicine, Indiana University Purdue University at Indianapolis, Indiana, USA 2 Ensenada Center for Scientific Research and Higher Education, Ensenada, Mexico 3 University of Virginia, Charlottesville, Virginia, USA 4 Institute Curie, Orsay, France 5 INSERM U1021, Orsay, France 6 CNRS UMR3347, Orsay, France 7 Universite Paris XI, Orsay, France Correspondence to: Theresa A. Guise, email: tguise@iu.edu Keywords: halofuginone, zoledronic acid, bone metastases, TGF-β, BMP Received: June 21, 2017 Accepted: August 04, 2017 Published: September 23, 2017 ABSTRACT More efficient therapies that target multiple molecular mechanisms are needed for the treatment of incurable bone metastases. Halofuginone is a plant alkaloid-derivative with antiangiogenic and antiproliferative effects. Here we demonstrate that halofuginone is an effective therapy for the treatment of bone metastases, through multiple actions that include inhibition of TGFβ and BMP-signaling. Halofuginone blocked TGF-β-signaling in MDA-MB-231 and PC3 cells showed by inhibition of TGF-β-induced Smad-reporter, phosphorylation of Smad-proteins, and expression of TGF-β-regulated metastatic genes. Halofuginone increased inhibitory Smad7-mRNA and reduced TGF-β-receptor II protein. Proline supplementation but not Smad7-knockdown reversed halofuginone-inhibition of TGF-β-signaling. Halofuginone also decreased BMP-signaling. Treatment of MDA-MB-231 and PC3 cells with halofuginone reduced the BMP-Smad-reporter (BRE) 4 , Smad1/5/8-phosphorylation and mRNA of the BMP-regulated gene Id-1. Halofuginone decreased immunostaining of phospho-Smad2/3 and phospho-Smad1/5/8 in cancer cells in vivo . Furthermore, halofuginone decreased tumor-take and growth of orthotopic-tumors. Mice with breast or prostate bone metastases treated with halofuginone had significantly less osteolysis than control mice. Combined treatment with halofuginone and zoledronic-acid significantly reduced osteolytic area more than either treatment alone. Thus, halofuginone reduces breast and prostate cancer bone metastases in mice and combined with treatment currently approved by the FDA is an effective treatment for this devastating complication of breast and prostate-cancer.

Objectives To determine if 2Flute (Stryker Corporation, Kalamazoo, MI) otologic burs improve the size, cellular content, and bone healing of autologous cortical bone grafts harvested during canal wall reconstruction (CWR) tympanomastoidectomy with mastoid obliteration. Study Design Institutional review board-approved prospective cohort study. Methods Human autologous cortical bone chips were harvested using various burs (4 and 6 mm diameter; multiflute, and 2Flute [Stryker Corporation]) from patients undergoing CWR tympanomastoidectomy for the treatment of chronic otitis media with cholesteatoma. Bone chip size, cell counts, cellular gene expression, and new bone formation were quantified. Results Bone chips were significantly larger when harvested with 2Flute (Stryker Corporation) bur compared to multiflute burs at both 6 mm diameter (113 ± 14 μm2 vs. 66 ± 8 μm2; P

// Trupti Trivedi 1, 2 , Yu Zheng 1 , Pierrick G.J. Fournier 2, 5 , Sreemala Murthy 2 , Sutha John 2 , Suzanne Schillo 1 , Colin R. Dunstan 3 , Khalid S. Mohammad 2 , Hong Zhou 1 , Markus J. Seibel 1, 4 , Theresa A. Guise 2 1 Bone Research Program, ANZAC Research Institute, University of Sydney, Sydney, Australia 2 Division of Endocrinology, Department of Medicine, Indiana University-Purdue University at Indianapolis, Indianapolis, Indiana, USA 3 Department of Biomedical Engineering, University of Sydney, Sydney, Australia 4 Department of Endocrinology and Metabolism, Concord Hospital, Concord, Sydney, Australia 5 Biomedical Innovation Department, Scientific Research and High Education Center from Ensenada (CICESE), Ensenada, Baja California, Mexico Correspondence to: Theresa A. Guise, email: tguise@iu.edu Markus J. Seibel, email: markus.seibel@sydney.edu.au Keywords: breast cancer, vitamin D, vitamin D receptor, ligand independent, bone metastasis Received: November 04, 2016 Accepted: February 15, 2017 Published: March 01, 2017 ABSTRACT Vitamin D has pleiotropic effects on multiple tissues, including malignant tumors. Vitamin D inhibits breast cancer growth through activation of the vitamin D receptor (VDR) and via classical nuclear signaling pathways. Here, we demonstrate that the VDR can also function in the absence of its ligand to control behaviour of human breast cancer cells both outside and within the bone microenvironment. Stable shRNA expression was used to knock down VDR expression in MCF-7 cells, generating two VDR knockdown clonal lines. In ligand-free culture, knockdown of VDR in MCF-7 cells significantly reduced proliferation and increased apoptosis, suggesting that the VDR plays a ligand-independent role in cancer cell growth. Implantation of these VDR knockdown cells into the mammary fat pad of nude mice resulted in reduced tumor growth in vivo compared with controls. In the intra-tibial xenograft model, VDR knockdown greatly reduced the ability of the cells to form tumors in the bone microenvironment. The in vitro growth of VDR knockdown cells was rescued by the expression of a mutant form of VDR which is unable to translocate to the nucleus and hence accumulates in the cytoplasm. Thus, our data indicate that in the absence of ligand, the VDR promotes breast cancer growth both in vitro and in vivo and that cytoplasmic accumulation of VDR is sufficient to produce this effect in vitro . This new mechanism of VDR action in breast cancer cells contrasts the known anti-proliferative nuclear actions of the VDR-vitamin D ligand complex.

Background and Hypothesis: Hyperglycemia is a major source of disease and morbidity among the adult population. Prior studies correlate long-term high fat diet (HFD) mediated hyperglycemia with bone fragility and muscle weakness. Furthermore, the mechanism driving hyperglycemia between sexes are unknown. Our group previously showed that HFDs induced insulin resistance in male mice and glucose intolerance in female mice. This establishes the need to study the impact of long-term HFDs on the bones and muscles using an older cohort of both male and female mice. For that, we hypothesized a long-term HFD mediated hyperglycemia will change bone and muscle structures and impair their functions in adult male and female mice. Experimental Design or Project Methods: 22-week C57Bl6 mice were fed either a HFD or low fat diet (LFD) for 25 weeks. After euthanasia, bones and muscles were harvested and evaluated using MicroCT, histology, and mechanical testing. Statistical analysis was performed using GraphPad Prism with p

// Laura E. Wright 1 , Ahmed A. Harhash 1 , Wende M. Kozlow 2 , David L. Waning 3 , Jenna N. Regan 1 , Yun She 1 , Sutha K. John 1 , Sreemala Murthy 1 , Maryla Niewolna 1 , Andrew R. Marks 4 , Khalid S. Mohammad 1 , Theresa A. Guise 1 1 Department of Medicine, Division of Endocrinology, Indiana University, Indianapolis, IN, USA 2 Department of Internal Medicine, Division of Endocrinology, University of Virginia, Charlottesville, VA, USA 3 Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, PA, USA 4 Department of Physiology, Columbia University, New York, NY, USA Correspondence to: Laura E. Wright, email: laewrig@iu.edu Keywords: breast cancer, bone, metastasis, aromatase inhibitor, skeletal muscle Received: October 20, 2016 Accepted: November 23, 2016 Published: December 25, 2016 ABSTRACT Aromatase inhibitors (AIs) cause muscle weakness, bone loss, and joint pain in up to half of cancer patients. Preclinical studies have demonstrated that increased osteoclastic bone resorption can impair muscle contractility and prime the bone microenvironment to accelerate metastatic growth. We hypothesized that AI-induced bone loss could increase breast cancer progression in bone and exacerbate muscle weakness associated with bone metastases. Female athymic nude mice underwent ovariectomy (OVX) or sham surgery and were treated with vehicle or AI (letrozole; Let). An OVX-Let group was then further treated with bisphosphonate (zoledronic acid; Zol). At week three, trabecular bone volume was measured and mice were inoculated with MDA-MB-231 cells into the cardiac ventricle and followed for progression of bone metastases. Five weeks after tumor cell inoculation, tumor-induced osteolytic lesion area was increased in OVX-Let mice and reduced in OVX-Let-Zol mice compared to sham-vehicle. Tumor burden in bone was increased in OVX-Let mice relative to sham-vehicle and OVX-Let-Zol mice. At the termination of the study, muscle-specific force of the extensor digitorum longus muscle was reduced in OVX-Let mice compared to sham-vehicle mice, however, the addition of Zol improved muscle function. In summary, AI treatment induced bone loss and skeletal muscle weakness, recapitulating effects observed in cancer patients. Prevention of AI-induced osteoclastic bone resorption using a bisphosphonate attenuated the development of breast cancer bone metastases and improved muscle function in mice. These findings highlight the bone microenvironment as a modulator of tumor growth locally and muscle function systemically.

Background and Hypothesis: Post-menopausal, estrogen-receptor positive breast cancer patients are treated with aromatase inhibitors (AIs) to limit tumor progression; however, this causes adverse musculoskeletal effects. Zoledronic acid (ZA) is prescribed to inhibit bone resorption. Mechanical signals, delivered via low intensity vibration (LIV), stimulate bone formation. We hypothesize that combining LIV with ZA will mitigate bone loss in a murine model of complete estrogen-deprivation more effectively than either treatment alone. Project Methods: 21-week-old C57BL/6 mice (n=20/group) were ovariectomized, receiving daily letrozole injections (OVX/AI) with LIV (OVX/AI+LIV), ZA (OVX/AI+ZA), LIV and ZA (OVX/AI+LIV/ZA) or underwent sham surgery with daily PBS (vehicle) injections (SH-OVX) for 22 weeks. Longitudinal dual energy X-ray absorptiometry (DEXA) and micro-computed tomography scans were analyzed for changes in body composition and bone microarchitecture, respectively. Results: OVX/AI reduced whole body (p< 0.0001) and lumbar spine (p

Increased fracture risk is commonly reported in cancer patients receiving radiotherapy, particularly at sites within the field of treatment. The direct and systemic effects of ionizing radiation on bone at a therapeutic dose are not well-characterized in clinically relevant animal models. Using 20-week-old male C57Bl/6 mice, effects of irradiation (right hindlimb; 2 Gy) on bone volume and microarchitecture were evaluated prospectively by microcomputed tomography and histomorphometry and compared to contralateral-shielded bone (left hindlimb) and non-irradiated control bone. One week postirradiation, trabecular bone volume declined in irradiated tibias (–22%; p

Muscle weakness and cachexia are significant paraneoplastic syndromes of many advanced cancers. Osteolytic bone metastases are common in advanced breast cancer and are a major contributor to decreased survival, performance, and quality of life for patients. Pathologic fracture caused by osteolytic cancer in bone (OCIB) leads to a significant (32%) increased risk of death compared to patients without fracture. Since muscle weakness is linked to risk of falls which are a major cause of fracture, we have investigated skeletal muscle response to OCIB. Here, we show that a syngeneic mouse model of OCIB (4T1 mammary tumor cells) leads to cachexia and skeletal muscle weakness associated with oxidation of the ryanodine receptor and calcium (Ca2+) release channel (RyR1). Muscle atrophy follows known pathways via both myostatin signaling and expression of muscle-specific ubiquitin ligases, atrogin-1 and MuRF1. We have identified a mechanism for skeletal muscle weakness due to increased oxidative stress on RyR1 via NAPDH oxidases [NADPH oxidase 2 (Nox2) and NADPH oxidase 4 (Nox4)]. In addition, SMAD3 phosphorylation is higher in muscle from tumor-bearing mice, a critical step in the intracellular signaling pathway that transmits TGFβ signaling to the nucleus. This is the first time that skeletal muscle weakness has been described in a syngeneic model of OCIB and represents a unique model system in which to study cachexia and changes in skeletal muscle.

Bone metastases occur in approximately 70% of metastatic breast cancer patients, often leading to skeletal injuries. Current treatments are mainly palliative and underscore the unmet clinical need for improved therapies. In this study, we provide preclinical evidence for an antimetastatic therapy based on targeting integrin β3 (β3), which is selectively induced on breast cancer cells in bone by the local bone microenvironment. In a preclinical model of breast cancer, β3 was strongly expressed on bone metastatic cancer cells, but not primary mammary tumors or visceral metastases. In tumor tissue from breast cancer patients, β3 was significantly elevated on bone metastases relative to primary tumors from the same patient (n = 42). Mechanistic investigations revealed that TGFβ signaling through SMAD2/SMAD3 was necessary for breast cancer induction of β3 within the bone. Using a micelle-based nanoparticle therapy that recognizes integrin αvβ3 (αvβ3-MPs of ∼12.5 nm), we demonstrated specific localization to breast cancer bone metastases in mice. Using this system for targeted delivery of the chemotherapeutic docetaxel, we showed that bone tumor burden could be reduced significantly with less bone destruction and less hepatotoxicity compared with equimolar doses of free docetaxel. Furthermore, mice treated with αvβ3-MP-docetaxel exhibited a significant decrease in bone-residing tumor cell proliferation compared with free docetaxel. Taken together, our results offer preclinical proof of concept for a method to enhance delivery of chemotherapeutics to breast cancer cells within the bone by exploiting their selective expression of integrin αvβ3 at that metastatic site. Cancer Res; 77(22); 6299–312. ©2017 AACR.

© 2017 Poor bone quality contributes to bone fragility in diabetes, aging, and osteogenesis imperfecta. However, the mechanisms controlling bone quality are not well understood, contributing to the current lack of strategies to diagnose or treat bone quality deficits. Transforming growth factor beta (TGF-β) signaling is a crucial mechanism known to regulate the material quality of bone, but its cellular target in this regulation is unknown. Studies showing that osteocytes directly remodel their perilacunar/canalicular matrix led us to hypothesize that TGF-β controls bone quality through perilacunar/canalicular remodeling (PLR). Using inhibitors and mice with an osteocyte-intrinsic defect in TGF-β signaling (TβRIIocy−/−), we show that TGF-β regulates PLR in a cell-intrinsic manner to control bone quality. Altogether, this study emphasizes that osteocytes are key in executing the biological control of bone quality through PLR, thereby highlighting the fundamental role of osteocyte-mediated PLR in bone homeostasis and fragility. Resistance to fracture requires healthy bone mass and quality. However, the cellular mechanisms regulating bone quality are unclear. Dole et al. show that osteocyte-intrinsic TGF-β signaling maintains bone quality through perilacunar/canalicular remodeling. Thus, osteocytes mediate perilacunar/canalicular remodeling and osteoclast-directed remodeling to cooperatively maintain bone quality and mass and prevent fragility.

Dysregulated transforming growth factor beta (TGF-β) signaling is associated with a spectrum of osseous defects as seen in Loeys-Dietz syndrome, Marfan syndrome, and Camurati-Engelmann disease. Intriguingly, neurofibromatosis type 1 (NF1) patients exhibit many of these characteristic skeletal features, including kyphoscoliosis, osteoporosis, tibial dysplasia, and pseudarthrosis; however, the molecular mechanisms mediating these phenotypes remain unclear. Here, we provide genetic and pharmacologic evidence that hyperactive TGF-β1 signaling pivotally underpins osseous defects in Nf1(flox/-) ;Col2.3Cre mice, a model which closely recapitulates the skeletal abnormalities found in the human disease. Compared to controls, we show that serum TGF-β1 levels are fivefold to sixfold increased both in Nf1(flox/-) ;Col2.3Cre mice and in a cohort of NF1 patients. Nf1-deficient osteoblasts, the principal source of TGF-β1 in bone, overexpress TGF-β1 in a gene dosage-dependent fashion. Moreover, Nf1-deficient osteoblasts and osteoclasts are hyperresponsive to TGF-β1 stimulation, potentiating osteoclast bone resorptive activity while inhibiting osteoblast differentiation. These cellular phenotypes are further accompanied by p21-Ras-dependent hyperactivation of the canonical TGF-β1-Smad pathway. Reexpression of the human, full-length neurofibromin guanosine triphosphatase (GTPase)-activating protein (GAP)-related domain (NF1 GRD) in primary Nf1-deficient osteoblast progenitors, attenuated TGF-β1 expression levels and reduced Smad phosphorylation in response to TGF-β1 stimulation. As an in vivo proof of principle, we demonstrate that administration of the TGF-β receptor 1 (TβRI) kinase inhibitor, SD-208, can rescue bone mass deficits and prevent tibial fracture nonunion in Nf1(flox/-) ;Col2.3Cre mice. In sum, these data demonstrate a pivotal role for hyperactive TGF-β1 signaling in the pathogenesis of NF1-associated osteoporosis and pseudarthrosis, thus implicating the TGF-β signaling pathway as a potential therapeutic target in the treatment of NF1 osseous defects that are refractory to current therapies.

Cancer-associated muscle dysfunction represents a deadly clinical problem, with ca. 80% mortality together with an increased toxicity from cancer treatment.. The normal bone remodeling might be disrupted by tumor cells that metastasize to bone in certain stages of cancer, which results in increased morbidity including muscle weakness. The reason for that muscle weakness might be attributed to the reduction on muscle mass or the reduction of muscle function. In fact, it has been demonstrated that in advanced cancers, it is probably caused by a combination of reductions, quantity and quality of muscle. This review focuses on the mechanisms that bone metastases promote skeletal muscle weakness.

Breast cancer is the most prevalent cancer among females worldwide leading to approximately 350,000 deaths each year. It has long been known that cancers preferentially metastasize to particular organs, and bone metastases occur in ~70% of patients with advanced breast cancer. Breast cancer bone metastases are predominantly osteolytic and accompanied by increased fracture risk, pain, nerve compression and hypercalcemia, causing severe morbidity. In the bone matrix, transforming growth factor-β (TGF-β) is one of the most abundant growth factors, which is released in active form upon tumor-induced osteoclastic bone resorption. TGF-β, in turn, stimulates bone metastatic tumor cells to secrete factors that further drive osteolytic bone destruction adjacent to the tumor. Thus, TGF-β is a crucial factor responsible for driving the feed-forward vicious cycle of cancer growth in bone. Moreover, TGF-β activates epithelial-to-mesenchymal transition, increases tumor cell invasiveness and angiogenesis and induces immunosuppression. Blocking the TGF-β signaling pathway to interrupt this vicious cycle between breast cancer and bone offers a promising target for therapeutic intervention to decrease skeletal metastasis. This review will describe the role of TGF-β in breast cancer and bone metastasis, and pre-clinical and clinical data will be evaluated for the potential use of TGF-β inhibitors in clinical practice to treat breast cancer bone metastases.

TGF-β derived from bone fuels melanoma bone metastases by inducing tumor secretion of prometastatic factors that act on bone cells to change the skeletal microenvironment. Halofuginone is a plant alkaloid derivative that blocks TGF-β signaling with antiangiogenic and antiproliferative properties. Here, we show for the first time that halofuginone therapy decreases development and progression of bone metastasis caused by melanoma cells through the inhibition of TGF-β signaling. Halofuginone treatment of human melanoma cells inhibited cell proliferation, phosphorylation of SMAD proteins in response to TGF-β, and TGF-β–induced SMAD-driven transcription. In addition, halofuginone reduced expression of TGF-β target genes that enhance bone metastases, including PTHrP, CTGF, CXCR4, and IL11. Also, cell apoptosis was increased in response to halofuginone. In nude mice inoculated with 1205Lu melanoma cells, a preventive protocol with halofuginone inhibited bone metastasis. The beneficial effects of halofuginone treatment were comparable with those observed with other anti–TGF-β strategies, including systemic administration of SD208, a small-molecule inhibitor of TGF-β receptor I kinase, or forced overexpression of Smad7, a negative regulator of TGF-β signaling. Furthermore, mice with established bone metastases treated with halofuginone had significantly less osteolysis than mice receiving placebo assessed by radiography. Thus, halofuginone is also effective in reducing the progression of melanoma bone metastases. Moreover, halofuginone treatment reduced melanoma metastasis to the brain, showing the potential of this novel treatment against cancer metastasis. Cancer Res; 72(23); 6247–56. ©2012 AACR.

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. Mol Cancer Res; 10(5); 597–604. ©2012 AACR.

Bone metastases are incurable. The bone microenvironment has always been a suspect for this clinical enigma, but the exact mechanisms have been unclear. In this issue of Cancer Cell, Zheng and colleagues provide evidence that chemotherapy itself induces chemoresistance of bone metastases, mediated by osteoblast Jagged1-induced tumor Notch signaling.

Prostate cancer (PCa), the most frequently diagnosed cancer in men, will have an estimated 26,730 deaths in the US during 2017. Predominantly, these deaths will be caused due to metastatic disease. PCa preferentially metastasizes to the bone undermining its structural integrity. PCa cells rely on androgens for survival and proliferation. Consequently, androgen deprivation therapy (ADT) is the first line of defense against advanced-stage PCa. However, ADT contributes to significant decreases in bone mass, predisposing the patient to an increased risk of fractures. Hence, there is a critical need for therapies that protect against bone metastatic disease as well as reverse ADT-induced bone loss in PCa patients. Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a direct target of the androgen receptor (AR) and is highly over-expressed in androgen-dependent and independent forms of PCa. In vitro studies have shown that its inhibition suppresses the growth and migration of PCa cells. We observed that CaMKK2 inhibition suppresses 3-D spheroid formation by the androgen-independent PCa cell line C4-2 but not by the androgen-dependent PC3 cells. However, the exact downstream mechanism by which CaMKK2 regulates PCa growth remains unknown. More recently, CaMKK2 was shown to play a role in both the anabolic and catabolic pathways of bone remodeling. Genetic ablation and pharmacological inhibition of CaMKK2 via STO-609 stimulates osteoblasts and suppresses osteoclasts and confers protection from age- and ovariectomy-induced bone loss in mice. Based on these preliminary data, we hypothesize that inhibition of a single target, CaMKK2, will result in the therapeutic alleviation of two chief complications in advanced-stage PCa, i.e., bone metastatic tumor burden and ADT-induced bone loss. We performed sham or bilateral orchiectomy (ORX) on pretreated (saline/STO-609) 5-week-old male athymic mice (n=15 per cohort). Tri-weekly intraperitoneal (i.p.) injections were continued for 12 weeks. Additionally, two weeks after surgery, sham and ORX mice were intra-tibially injected with C4-2B cells. Micro-computed tomography analyses indicated a prevention of ORX-induced bone loss in STO-609 treated mice compared to saline treated controls (3-fold, p Citation Format: Ushashi Dadwal, Justin Williams, Austin Pucylowski, Eric Chang, Khalid Mohammad, Theresa Guise, Uma Sankar. CaMKK2 inhibition as a "dual-hit" strategy against ADT-induced osteoporosis and bone-metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3987.

Endoxifen, the predominant CYP2D6 metabolite of the selective estrogen receptor modulator (SERM) tamoxifen, is currently being developed as a novel anti-estrogen therapy for the treatment of estrogen receptor (ER)+ breast cancer. Genetic polymorphism of CYP2D6 is a predictor of response to tamoxifen in patients, implicating endoxifen as one of the most active and relevant tamoxifen metabolites. Breast cancer patients treated with adjuvant endocrine therapies including aromatase inhibitors (AIs) and SERMs often report unmanageable musculoskeletal toxicities that can result in treatment discontinuation. While ER binding affinity and anti-tumor effects have been established in preclinical models, the effects of endoxifen on the musculoskeletal system are not fully known. Twenty-week female C57BL/6 mice underwent sham surgery or ovariectomy (OVX) and were treated daily with vehicle, the AI letrozole, or the SERM endoxifen. Body composition was assessed prospectively by DXA. Bone indices and marrow adipose tissue volume were measured by μCT and muscle contractility of the extensor digitorum longus (EDL) was measured ex vivo. After eight weeks, trabecular bone volume fraction (BV/TV) decreased by 50% in OVX-vehicle and OVX-letrozole mice, whereas BV/TV increased threefold in endoxifen mice relative to sham-vehicle. Despite the presence of significantly more trabecular bone following endoxifen treatment, cortical bone analyses revealed impaired periosteal and endosteal expansion of bone, resulting in reduced polar moment of inertia, which is a measure of resistance to fracture. Uterine weight increased significantly in endoxifen-treated mice relative to all OVX groups, similar to what is observed with tamoxifen treatment. Peripheral body fat, bone marrow adipose tissue, and circulating leptin were reduced by endoxifen, suggesting that the drug may elicit positive systemic metabolic effects. At the termination of the study, muscle-specific force was reduced in OVX-endoxifen mice relative to sham-vehicle, OVX-vehicle, and OVX-AI mice, despite no change in muscle mass. While endoxifen shows promise as a potent anti-estrogen therapy for the treatment of ER+ breast cancer, it may be important to monitor patients for endometrial proliferation, morphological changes in bone that increase fracture risk, and for musculoskeletal side effects that could reduce drug compliance. Citation Format: Laura Wright, Khalid Mohammad, Theresa Guise. The anti-estrogen endoxifen altered bone morphology and reduced muscle function in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1808.

Bone is the most common site for metastasis of advanced prostate cancers. Once housed in the skeleton, tumors are incurable and cause protracted morbidity, and bone metastases may contribute to mortality through unknown mechanisms. Bone provides a unique microenvironment whose local interactions with tumor cells offer novel targets for therapeutic interventions. Many standard cancer treatments cause bone loss, which may aggravate skeletal metastases, although this is preventable with approved agents. Improved bonetargeted treatments can decrease the serious skeletal morbidities associated with metastatic prostate cancer and may in the future improve overall survival. The development of such treatments requires preclinical evaluation in animal models of prostate cancer growth in bone.

Certain solid tumors metastasize to bone and cause osteolysis and abnormal new bone formation. The respective phenotypes of dysregulated bone destruction and bone formation represent two ends of a spectrum, and most patients will have evidence of both. The mechanisms responsible for tumor growth in bone are complex and involve tumor stimulation of the osteoclast and the osteoblast as well as the response of the bone microenvironment. Furthermore, factors that increase bone resorption, independent of tumor, such as sex steroid deficiency, may contribute to this vicious cycle of tumor growth in bone. This article discusses mechanisms and therapeutic implications of osteolytic and osteoblastic bone metastases.

Hypercalcemia associated with malignancy is an old clinical problem, first described by Fuller Albright more than 50 years ago. It can cause significantmorbidity in patients with cancer and is associated with a poor prognosis in most patients. Thus, it is important to understand the pathophysiology to properly diagnosis and treat this important clinical syndrome. The accompanying article by Goldner presents a comprehensive review of this topic, which includes the latest addition to our therapeutic armamentarium to lower pathologic increases in serum calcium concentration: the receptor activator of nuclear factor k B–ligand antibody denosumab. Goldnermakes several important points in this review. First, the clinician must determine if the hypercalcemia is indeed a result of malignancy, rather than just associated with a nonmalignant cause of hypercalcemia such as primary hyperparathyroidism. After this has been established, treatment is directed against the cancer as well as thehypercalcemia. Tumor-produced systemic factors, such as parathyroid hormone–related peptide (PTHrP) or 1,25dihydroxyvitaminD, or locally active factors in bone, such as interleukin-6 or tumor necrosis factor-a, disrupt normal calcium homeostasis by excessive stimulation of osteoclastic bone resorption, in addition to lesser effects, to increase renal tubular reabsorption of calcium to result in hypercalcemia. As such, the mainstay of supportive care is to block pathologic osteoclast activity and maintain adequate hydration. For many years, this therapy consisted of bisphosphonates and fluid hydration, and for the most part was effective, particularly with the advent of the potent bisphosphonate zoledronic acid. As therapy evolved to include the most potent bisphosphonates, our understanding of the pathophysiology also evolved. The discovery thatmost tumor osteolytic factors, either humoral or local, stimulated bone resorption via receptor activator of nuclear factor k B–ligand led to more potent therapy to block osteoclastic bone resorption with the monoclonal antibody denosumab directed against this powerful stimulator of osteoclast formation and resorption. Indeed, recent reports indicate that denosumab is effective to reduce serum calcium concentration in the setting of bisphosphonate resistance and is more effective than zoledronic acid to prevent hypercalcemia associated with bone metastases. One downside to this new and more potent treatment is the increased risk of hypocalcemia. Thus, calcium should be closely monitored with the use of denosumab. Furthermore, because the studies describing denosumab use are small or retrospective analyses, further clinical studies using denosumab in the setting of hypercalcemia of malignancy are indicated. A future molecular target for the treatment of hypercalcemiaofmalignancy involvesPTHrPrelated antibodies. In an animal model, PTHrP antibodies corrected hypercalcemia and also suppressed the release of cytokines associated with malignancy. Taken together, insight into the pathogenesis of hypercalcemia ofmalignancy has resulted in new and better therapy for an old but difficult clinical problem.

Osteoblastic bone metastases are common in prostate and breast cancer patients, but mechanisms by which tumor cells stimulate new bone formation are unclear. We identified three breast cancer cell lines that cause osteoblastic metastases in a mouse model and secrete endothelin-1. Tumor-produced endothelin-1 stimulates new bone formation in vitro and osteoblastic metastases in vivo via the endothelin A receptor. Treatment with an orally active endothelin A receptor antagonist dramatically decreased bone metastases and tumor burden in mice inoculated with ZR-75-1 cells. Tumor-produced endothelin-1 may have a major role in the establishment of osteoblastic bone metastases, and endothelin A receptor blockade represents effective treatment.

BACKGROUND Certain solid tumors metastasize to bone and cause an osteoblastic response. The mechanisms by which tumor cells stimulate this new bone formation are not completely understood. METHODS The authors identified three breast cancer lines that cause osteoblastic metastases in female nude mice and provided evidence that tumor-produced endothelin-1 (ET-1) mediates the osteoblastic response. RESULTS Tumor conditioned media, as well as exogenous ET-1, stimulated osteoblast proliferation and new bone formation in cultures of mouse calvariae. These effects were blocked by antagonists of the endothelin A (ETA), but not ETB, receptors. Mice inoculated with the ZR-75-1 breast cancer line and treated with a selective ETA receptor antagonist (ABT-627) had significantly fewer osteoblastic bone metastases and less tumor burden compared with untreated mice. In contrast, there was no effect of ABT-627 on osteolytic bone metastases caused by ET-1-negative breast cancer, MDA-MB-231. ABT-627 had no effect on growth in vitro or at the orthotopic site of ZR-75-1 or MDA-MB-231 cells. CONCLUSIONS Collectively, the data suggested that tumor-produced ET-1 mediates osteoblastic bone metastases by stimulating osteoblast proliferation and new bone formation. ETA receptor blockade may be useful for prevention and the treatment of osteoblastic bone metastases due to breast or prostate cancer. Cancer 2003;97(3 Suppl):779–84. © 2003 American Cancer Society. DOI 10.1002/cncr.11129

Despite the high prevalence and significant morbidity of spinal anomalies in neurofibromatosis type 1 (NF1), the pathogenesis of these defects remains largely unknown. Here, we present two murine models: Nf1flox/-;PeriCre and Nf1flox/-;Col.2.3Cre mice, which recapitulate spinal deformities seen in the human disease. Dynamic histomorphometry and microtomographic studies show recalcitrant bone remodeling and distorted bone microarchitecture within the vertebral spine of Nf1flox/-;PeriCre and Nf1flox/-;Col2.3Cre mice, with analogous histological features present in a human patient with dystrophic scoliosis. Intriguingly, 36-60% of Nf1flox/-;PeriCre and Nf1flox/-;Col2.3Cre mice exhibit segmental vertebral fusion anomalies with boney obliteration of the intervertebral disc (IVD). While analogous findings have not yet been reported in the NF1 patient population, we herein present two case reports of IVD defects and interarticular vertebral fusion in patients with NF1. Collectively, these data provide novel insights regarding the pathophysiology of dystrophic spinal anomalies in NF1, and provide impetus for future radiographic analyses of larger patient cohorts to determine whether IVD and vertebral fusion defects may have been previously overlooked or underreported in the NF1 patient population.

Study Design. In vivo experiments to develop a rat spine single metastasis model by using human breast cancer cells. Objective. To study the survival and tumorigenesis of the human breast cancer cells after transplantation to vertebral body (VB) by intraosseous injection as a model for therapeutic studies of spine metastatic tumor. Summary of Background Data. VBs are the most common bones involved in the metastases of breast cancer. To develop experimental therapeutics requires an appropriate animal model. Moreover, it is also important to establish accurate and sensitive detection methods for the evaluation. Methods. MDA-MB-231 human breast cancer cells were injected into 3-week-old female athymic rats. The tumorigenesis was assayed with quantitative in vivo bioluminescence (IVIS), microcomputed tomography (micro-CT), quantitative CT (qCT), micro position emission tomography (micro-PET), and histologic studies. Results. A spine single metastasis model of human breast cancer was successfully developed in rats. The IVIS signal intensity from the cancer cells increased after 2 weeks. Signal from the tumor in spine can be detected by micro-PET at day 1. The signal intensity decreased after 1 week and then recovered and continually increased afterwards. Bone destruction was demonstrated in the qCT and micro-CT images. However, both qCT and micro-CT found that the bone density in the cancer cell-injected VB increased before the appearance of osteolysis. The growth of tumor and the reaction of bone in the VB were observed simultaneously by histology. Conclusion. A spine single metastasis model was developed by injection of human breast cancer cells into the VB of athymic rats. This is the first report of quantitative evaluation with micro-PET in a spine metastasis model. In addition, the detection of osteogenesis after the introduction of MDA-MB-231 cells in vivo is a novel observation.

Background Most patients with advanced breast cancer develop bone metastases, which cause pain, hypercalcemia, fractures, nerve compression and paralysis. Chemotherapy causes further bone loss, and bone-specific treatments are only palliative. Multiple tumor-secreted factors act on the bone microenvironment to drive a feed-forward cycle of tumor growth. Effective treatment requires inhibiting upstream regulators of groups of prometastatic factors. Two central regulators are hypoxia and transforming growth factor (TGF)- β. We asked whether hypoxia (via HIF-1α) and TGF-β signaling promote bone metastases independently or synergistically, and we tested molecular versus pharmacological inhibition strategies in an animal model. Methodology/Principal Findings We analyzed interactions between HIF-1α and TGF-β pathways in MDA-MB-231 breast cancer cells. Only vascular endothelial growth factor (VEGF) and the CXC chemokine receptor 4 (CXCR4), of 16 genes tested, were additively increased by both TGF-β and hypoxia, with effects on the proximal promoters. We inhibited HIF-1α and TGF-β pathways in tumor cells by shRNA and dominant negative receptor approaches. Inhibition of either pathway decreased bone metastasis, with no further effect of double blockade. We tested pharmacologic inhibitors of the pathways, which target both the tumor and the bone microenvironment. Unlike molecular blockade, combined drug treatment decreased bone metastases more than either alone, with effects on bone to decrease osteoclastic bone resorption and increase osteoblast activity, in addition to actions on tumor cells. Conclusions/Significance Hypoxia and TGF-β signaling in parallel drive tumor bone metastases and regulate a common set of tumor genes. In contrast, small molecule inhibitors, by acting on both tumor cells and the bone microenvironment, additively decrease tumor burden, while improving skeletal quality. Our studies suggest that inhibitors of HIF-1α and TGF-β may improve treatment of bone metastases and increase survival.

Mammals are able to regrow the tips of amputated fingers and toes. However, regrowth is limited to regions covered by, and is dependent upon, the presence of the nail organ. If the nail organ is responsible for bone growth in digit-tips, we reasoned that transplanted nail organ might also be able to induce outgrowth from other levels of the digit. Partial nail organ has been transplanted to amputated proximal phalanges of young rats. To date, six transplants have successfully produced outgrowth of nail. New bone growth, not seen in control amputated digits, was documented by x-ray and by alizarin red and calcein injections to be directed toward implanted nail organ. These results support an inductive role for nail organ epithelium in bone growth after amputation and provide encouragement for attempts to enhance a positive outcome after appendage amputation.

Melanoma has a propensity to metastasize to bone, where it is exposed to high concentrations of transforming growth factor-β (TGF-β). Because TGF-β promotes bone metastases from other solid tumors, such as breast cancer, we tested the role of TGF-β in melanoma metastases to bone. 1205Lu melanoma cells, stably transfected to overexpress the natural TGF-β/Smad signaling inhibitor Smad7, were studied in an experimental model of bone metastasis whereby tumor cells are inoculated into the left cardiac ventricle of nude mice. All mice bearing parental and mock-transfected 1205Lu cells developed osteolytic bone metastases 5 weeks post-tumor inoculation. Mice bearing 1205Lu-Smad7 tumors had significantly less osteolysis on radiographs and longer survival compared with parental and mock-transfected 1205Lu mice. To determine if the reduced bone metastases observed in mice bearing 1205Lu-Smad7 clones was due to reduced expression of TGF-β target genes known to enhance metastases to bone from breast cancer cells, we analyzed gene expression of osteolytic factors, parathyroid hormone-related protein (PTHrP) and interleukin-11 (IL-11), the chemotactic receptor CXCR4, and osteopontin in 1205Lu cells. Quantitative reverse transcription-PCR analysis indicated that PTHrP, IL-11, CXCR4, and osteopontin mRNA steady-state levels were robustly increased in response to TGF-β and that Smad7 and the TβRI small-molecule inhibitor, SB431542, prevented such induction. In addition, 1205Lu-Smad7 bone metastases expressed significantly lower levels of IL-11, connective tissue growth factor, and PTHrP. These data suggest that TGF-β promotes osteolytic bone metastases due to melanoma by stimulating the expression of prometastatic factors via the Smad pathway. Blockade of TGF-β signaling may be an effective treatment for melanoma metastasis to bone. [Cancer Res 2007;67(5):2317–24]

Since their isolation in 1988, endothelins have emerged as modulators of many functions including vasomotor tone, hormone production and cell proliferation. Endothelins and their receptors are expressed by many tissues, thus, it is no surprise that endothelins play an important role in the normal physiological functions and pathological states. Abundant evidence implicates a role for endothelins in cancer. This review will present evidence that endothelins play a major role in the process of cancer metastases to bone.

Certain solid tumors metastasize to bone, causing an osteoblastic response. The mechanisms by which tumor cells stimulate this new bone formation are not understood completely. We identified three breast cancer lines that cause osteoblastic metastases in female nude mice and provide evidence that tumor-produced endothelin-1 (ET-1) mediates the osteoblastic response. Tumor-conditioned media and exogenous ET-1 stimulated osteoblast proliferation and new bone formation in cultures of calvarias from mice. These effects were blocked by endothelin A (ETA) but not by ETB receptor antagonists. Mice inoculated with the ZR-75-1 breast cancer line and treated with a selective ETA receptor antagonist (ABT-627) had significantly fewer osteoblastic bone metastases and less tumor burden compared with untreated mice. In contrast, there was no effect of ABT-627 on osteolytic bone metastases caused by ET-1-negative breast cancer, MDA-MB-231. ABT-627 had no effect on cell growth in vitro or at the orthotopic site (mammary fat pad) of ZR-75-1, or MDA-MB-231 cells. Collectively, the data suggest that tumor-produced ET-1 mediates osteoblastic bone metastases by stimulating osteoblast proliferation and new bone formation. Endothelin A receptor blockade may be useful for the prevention and treatment of osteoblastic bone metastases attributable to breast or prostate cancer.

Rapid tumor growth results in tumor hypoxia and the induction of key mediators of angiogenesis including VEGF and MET. Cells in the bone microenvironment, including osteoblasts and osteoclasts, express MET and VEGFRs and respond to HGF and VEGF. Cabozantinib (cabo) is an inhibitor of tyrosine kinases including MET, VEGFR2, and RET. Cabo treatment in preclinical models results in tumor regression and blockade of tumor invasiveness and metastasis, and has shown clinical activity in patients with castration-resistant prostate cancer and breast cancer tumors with bone metastases. To elucidate the mechanisms underlying some of these clinical observations, the effects of cabo were studied in a human breast cancer bone xenograft and in a non-tumor bearing model. Female nude mice were inoculated with MDA-MB-231 cells into the left cardiac ventricle and treated with cabo (10 or 60 mg/kg/day). Treatment was initiated 13 days after tumor inoculation when osteolytic lesions were detectable on x-ray, and continued for 11 days. In a non-tumor model, female nude mice (∼5 weeks old) were treated with similar doses for 28 days. Tumor-bearing mice treated with cabo 60 mg/kg showed a reduction in osteolytic lesion area as measured by x-ray (p Note: This abstract was not presented at the meeting. Citation Format: Khalid S. Mohammad, Ahmed Harhash, Sutha K. John, Xianghong Peng, Maria Niewolna, Sreemala Murthy, A. Douglas Laird, Dana T. Aftab, Theresa A. Guise. Effects of cabozantinib on breast cancer bone metastases, overall survival, and bone mass in a mouse model. [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 733. doi:10.1158/1538-7445.AM2014-733

The term bone regeneration is used either to describe bone repair or, more accurately, to describe bone formation in sites at which bone would not normally be expected to reform such as avulsion or amputation. Induced regeneration of bone is becoming one of the major success stories of biomedical research. Keywords: regeneration; bone replacement; growth factors; osteogenesis; bone remodelling; bone growth

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

SummaryTransforming growth factor-β (TGF-β) regulates the expression of genes supporting breast cancer cells in bone, but little is known about prostate cancer bone metastases and TGF-β. Our study reveals that the TGFBR1 inhibitor SD208 effectively reduces prostate cancer bone metastases. TGF-β upregulates in prostate cancer cells a set of genes associated with cancer aggressiveness and bone metastases, and the most upregulated gene was PMEPA1. In patients, PMEPA1 expression decreased in metastatic prostate cancer and low Pmepa1 correlated with decreased metastasis-free survival. Only membrane-anchored isoforms of PMEPA1 interacted with R-SMADs and ubiquitin ligases, blocking TGF-β signaling independently of the proteasome. Interrupting this negative feedback loop by PMEPA1 knockdown increased prometastatic gene expression and bone metastases in a mouse prostate cancer model.

Introduction Adrenomedullin (AM) is secreted by breast cancer cells and increased by hypoxia. It is a multifunctional peptide that stimulates angiogenesis and proliferation. The peptide is also a potent paracrine stimulator of osteoblasts and bone formation, suggesting a role in skeletal metastases—a major site of treatment-refractory tumor growth in patients with advanced disease. Methods The role of adrenomedullin in bone metastases was tested by stable overexpression in MDA-MB-231 breast cancer cells, which cause osteolytic bone metastases in a standard animal model. Cells with fivefold increased expression of AM were characterized in vitro, inoculated into immunodeficient mice and compared for their ability to form bone metastases versus control subclones. Bone destruction was monitored by X-ray, and tumor burden and osteoclast numbers were determined by quantitative histomorphometry. The effects of AM overexpression on tumor growth and angiogenesis in the mammary fat pad were determined. The effects of AM peptide on osteoclast-like multinucleated cell formation were tested in vitro. A small-molecule AM antagonist was tested for its effects on AM-stimulated ex vivo bone cell cultures and co-cultures with tumor cells, where responses of tumor and bone were distinguished by species-specific real-time PCR. Results Overexpression of AM mRNA did not alter cell proliferation in vitro, expression of tumor-secreted factors or cell cycle progression. AM-overexpressing cells caused osteolytic bone metastases to develop more rapidly, which was accompanied by decreased survival. In the mammary fat pad, tumors grew more rapidly with unchanged blood vessel formation. Tumor growth in the bone was also more rapid, and osteoclasts were increased. AM peptide potently stimulated bone cultures ex vivo; responses that were blocked by small-molecule adrenomedullin antagonists in the absence of cellular toxicity. Antagonist treatment dramatically suppressed tumor growth in bone and decreased markers of osteoclast activity. Conclusions The results identify AM as a target for therapeutic intervention against bone metastases. Adrenomedullin potentiates osteolytic responses in bone to metastatic breast cancer cells. Small-molecule antagonists can effectively block bone-mediated responses to tumor-secreted adrenomedullin, and such agents warrant development for testing in vivo. Electronic supplementary material The online version of this article (doi:10.1186/s13058-014-0458-y) contains supplementary material, which is available to authorized users.