Tumor-expressed adrenomedullin accelerates breast cancer bone metastasis.

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.

Regulation of postnatal trabecular bone formation by the osteoblast endothelin A receptor.

Endothelin-1 (ET-1) is a potent vasoconstrictor that also stimulates cells in the osteoblast lineage by binding to the endothelin A receptor (ETAR). ET-1 ligand is widely secreted, particularly by the vasculature. However, the contributions of ETAR signaling to adult bone homeostasis have not been defined. ETAR was inactivated in osteoblasts by crossing ETAR-floxed and osteocalcin-Cre mice. Histomorphometric analyses were performed on 4-, 8-, and 12-week-old osteoblast-targeted ETAR knockout (KO) and wild-type (WT) male and female mice. Tibial trabecular bone volume was significantly lower from 12 weeks in KO versus WT mice in both males and females. Bone-formation rate, osteoblast density, and in vitro osteoblast differentiation were reduced by targeted inactivation of ETAR. A separate longitudinal analysis was performed between 8 and 64 weeks to examine the effect of aging and castration on bone metabolism in ETAR KO mice. Hypogonadism did not change the rate of bone accrual in WT or KO females. However, eugonadal KO males had a significantly larger increase in tibial and femoral bone acquisition than WT mice. Male mice castrated at 8 weeks of age showed the reverse: KO mice had reduced rates of tibial and femoral BMD acquisition compared with WT mice. In vitro, ET-1 increased osteoblast proliferation, survival, and differentiation. Dihydrotestosterone also increased osteoblast differentiation using a mechanism distinct from the actions of ET-1. These results demonstrate that endothelin signaling in osteoblasts is an important regulator of postnatal trabecular bone remodeling and a modulator of androgen effects on bone.

TGF-beta-RI kinase inhibitor SD-208 reduces the development and progression of melanoma bone metastases.

Melanoma often metastasizes to bone where it is exposed to high concentrations of TGF-ß. Constitutive Smad signaling occurs in human melanoma. Because TGF-ß promotes metastases to bone by several types of solid tumors including breast cancer, we hypothesized that pharmacologic blockade of the TGF-ß signaling pathway may interfere with the capacity of melanoma cells to metastasize to bone. In this study, we tested the effect of a small molecule inhibitor of TGF-ß receptor I kinase (TßRI), SD-208, on various parameters affecting the development and progression of melanoma, both in vitro and in a mouse model of human melanoma bone metastasis. In melanoma cell lines, SD-208 blocked TGF-ß induction of Smad3 phosphorylation, Smad3/4-specific transcription, Matrigel invasion and expression of the TGF-ß target genes PTHrP, IL-11, CTGF, and RUNX2. To assess effects of SD-208 on melanoma development and metastasis, nude mice were inoculated with 1205Lu melanoma cells into the left cardiac ventricle and drug was administered by oral gavage on prevention or treatment protocols. SD-208 (60 mg/kg/d), started 2 days before tumor inoculation prevented the development of osteolytic bone metastases compared with vehicle. In mice with established bone metastases, the size of osteolytic lesions was significantly reduced after 4 weeks treatment with SD-208 compared with vehicle-treated mice. Our results demonstrate that therapeutic targeting of TGF-ß may prevent the development of melanoma bone metastases and decrease the progression of established osteolytic lesions.

Hypoxia and TGF-beta drive breast cancer bone metastases through parallel signaling pathways in tumor cells and the bone microenvironment.

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)- beta. We asked whether hypoxia (via HIF-1alpha) and TGF-beta 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-1alpha and TGF-beta 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-beta and hypoxia, with effects on the proximal promoters. We inhibited HIF-1alpha and TGF-beta 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-beta 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-1alpha and TGF-beta may improve treatment of bone metastases and increase survival.

Pharmacologic inhibition of the TGF-beta type I receptor kinase has anabolic and anti-catabolic effects on bone.

During development, growth factors and hormones cooperate to establish the unique sizes, shapes and material properties of individual bones. Among these, TGF-beta has been shown to developmentally regulate bone mass and bone matrix properties. However, the mechanisms that control postnatal skeletal integrity in a dynamic biological and mechanical environment are distinct from those that regulate bone development. In addition, despite advances in understanding the roles of TGF-beta signaling in osteoblasts and osteoclasts, the net effects of altered postnatal TGF-beta signaling on bone remain unclear. To examine the role of TGF-beta in the maintenance of the postnatal skeleton, we evaluated the effects of pharmacological inhibition of the TGF-beta type I receptor (TbetaRI) kinase on bone mass, architecture and material properties. Inhibition of TbetaRI function increased bone mass and multiple aspects of bone quality, including trabecular bone architecture and macro-mechanical behavior of vertebral bone. TbetaRI inhibitors achieved these effects by increasing osteoblast differentiation and bone formation, while reducing osteoclast differentiation and bone resorption. Furthermore, they induced the expression of Runx2 and EphB4, which promote osteoblast differentiation, and ephrinB2, which antagonizes osteoclast differentiation. Through these anabolic and anti-catabolic effects, TbetaRI inhibitors coordinate changes in multiple bone parameters, including bone mass, architecture, matrix mineral concentration and material properties, that collectively increase bone fracture resistance. Therefore, TbetaRI inhibitors may be effective in treating conditions of skeletal fragility.