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 TGFß and BMP-signaling. Halofuginone blocked TGF-ß-signaling in MDA-MB-231 and PC3 cells showed by inhibition of TGF-ß-induced Smad-reporter, phosphorylation of Smad-proteins, and expression of TGF-ß-regulated metastatic genes. Halofuginone increased inhibitory Smad7-mRNA and reduced TGF-ß-receptor II protein. Proline supplementation but not Smad7-knockdown reversed halofuginone-inhibition of TGF-ß-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.
TGF-ß 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-ß 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-ß signaling. Halofuginone treatment of human melanoma cells inhibited cell proliferation, phosphorylation of SMAD proteins in response to TGF-ß, and TGF-ß-induced SMAD-driven transcription. In addition, halofuginone reduced expression of TGF-ß 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 1205 Lu 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-ß strategies, including systemic administration of SD208, a small-molecule inhibitor of TGF-ß receptor I kinase, or forced overexpression of Smad7, a negative regulator of TGF-ß 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.
Antineoplastic Agents/pharmacology , Bone Neoplasms/prevention & control , Bone Neoplasms/secondary , Melanoma/drug therapy , Piperidines/pharmacology , Quinazolinones/pharmacology , Animals , Apoptosis/drug effects , Bone Neoplasms/metabolism , Cell Growth Processes/drug effects , Cell Line, Tumor , Disease Progression , Female , Gene Expression , Humans , Melanoma/metabolism , Melanoma/pathology , Melanoma/secondary , Mice , Mice, Nude , Signal Transduction , Xenograft Model Antitumor Assays
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.
Bone Neoplasms/prevention & control , Melanoma/pathology , Protein Kinase Inhibitors/pharmacology , Pteridines/pharmacology , Receptors, Transforming Growth Factor beta/antagonists & inhibitors , Animals , Base Sequence , Bone Neoplasms/secondary , Cell Line, Tumor , DNA Primers , Disease Models, Animal , Disease Progression , Gene Expression Profiling , Humans , Mice , Mice, Nude , Neoplasm Invasiveness , Phosphorylation , Smad Proteins/metabolism
