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    Research Grants Awarded

    Halofuginone Inhibition Of Tgf-Beta Signaling:Treatment For Bone Metastases

    Grant Mechanism:
    Postdoctoral Fellowships

    Scientific Abstract:
    HALOFUGINONE INHIBITION OF TGF-BETA SIGNALING: TREATMENT FOR BONE METASTASES BACKGROUND: Breast cancer commonly metastasizes to bone, causing pain and fracture. Transforming growth factor-beta (TGF-beta) is a major bone-derived factor responsible for driving the vicious cycle of breast cancer metastasis by increasing tumor production of osteolytic and pro-metastatic factors, such as parathyroid hormone-related protein (PTHrP), interleukin 11 (IL-11), vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF) and the homing receptor CXCR4, which stimulate bone destruction, tumor homing and growth and angiogenesis. Although TGF-beta is a suppressor of tumor development in normal breast epithelial cells, it becomes a pro-metatastic factor for advanced cancers, where it activates the epithelial-mesenchymal transition, tumor cell invasion, angiogenesis and immunosuppression. TGF-beta binds to a heteromeric complex of transmembrane serine/threonine kinases, the type I and type II receptors (TBRI and TBRII), which phosphorylate the intracellular signaling mediators Smad2 and Smad3. Active Smad2/3 complex then binds Smad4 and translocates to the nucleus, where it regulates gene transcription. TGF-beta also induces inhibitory Smad7, which can interact with TBRI and prevent the phosphorylation of the Smad2/3. Blocking TGF-beta signaling in cancer cells with ligand-neutralizing antibodies, dominant-negative receptors, small molecule kinase inhibitors or Smad7 overexpression all inhibit tumor growth or metastasis in animal models, but none of these inhibitory strategies has reached the clinic. HALOFUGINONE (Hfg) is a synthetic derivative of the plant alkaloid febrifugine. It suppresses the phosphorylation and activation of Smad2 and Smad3 by inducting of Smad7. Overexpression of inhibitory Smad7 has been associated with a reduction on invasive capacity in vitro and anchorage-independent growth, and delays subcutaneous tumor growth in nude mice, while the Principal Investigator has shown that Smad7 overexpression decreased melanoma bone metastasis. The role of TGF-beta in angiogenesis and metastases suggests that halofuginone should be an effective treatment for bone metastases. Hfg is active orally and by intraperitoneal injection. Effects of the drug on bone or tumors in bone have not been reported, and this research will determine the efficacy of Hfg against breast cancer bone metastases. HYPOTHESIS and OBJECTIVES: Transforming growth factor-beta (TGF-beta) is a major bone-derived factor responsible for driving a vicious cycle of breast cancer metastasis. Hfg is a natural product derivative that inhibits TGF-beta signaling by increasing expression of inhibitory Smad7. In this study we have two goals: to test the efficacy of Hfg against breast cancer bone metastases and to elucidate the molecular mechanisms responsible for the inhibition of TGF-beta signaling Hfg. WE HYPOTHESIZE THAT HALOFUGINONE WILL EFFECTIVELY INHIBIT BREAST CANCER METASTASES TO BONE IN A PRECLINICAL ANIMAL MODEL. SPECIFIC AIM 1: TO TEST THE ABILITY OF HFG TO DECREASE BREAST CANCER BONE METASTASES. Mice receiving breast cancer cells will be treated with drug and changes in tumor burden in bone analyzed. Female BALB/c nude mice will be inoculated via the left cardiac ventricle with MDA-MB-231 cells, a model that results in rapid and reproducible metastases to bone. Hfg will be administered throughout the study. SPECIFIC AIM 2: TO TEST IF THE EFFECTS OF HFG AGAINST TUMOR ARE SPECIFIC TO BONE. Breast cancer cells will be grown as mammary fat pad tumors and the effects of Hfg treatment on tumor burden compared to the effects in bone from Aim 1. SPECIFIC AIM 3: TO DETERMINE IF THE ACTIONS OF HFG ARE THROUGH INHIBITION OF THE TGF-BETA SIGNALING PATHWAY. The effects of Hfg treatment will be compared, as in Aim 1, to those caused by a small molecule inhibitor of the type I TGF-beta receptor kinase activity, SD-208. Results from experiments in vivo will be compared to changes seen in vitro on the activity of Smads 2, 3 and 7. The effects of Hfg on TGF-beta signaling and TGF-beta-regulated genes (such as PTHrP, IL11, CTGF, VEGF and CXCR4) in bone metastases will also be evaluated. POTENTIAL OUTCOMES AND BENEFITS OF THE RESEARCH TGF-beta is a multifunctional cytokine with an established pro-metastatic role in advanced cancers. Blockade of signaling from the TGF-beta receptor decreases bone metastases. Hfg inhibits TGF-beta-dependent phosphorylation of Smad2/3 and induces Smad7, suggesting that it could be an effective specific treatment for bone metastases, which could be rapidly translated to the clinic. Extensive animal use indicates that the agent is safe and effective in vivo. The agent is being taken into Phase II trails in patients with scleroderma. The work proposed will provide the proof of principle and mechanistic data to determine if the agent should be tested for efficacy against bone metastases in patients with advanced breast cancer. The work will permit rapid translation of Hfg from laboratory to clinic.

    Lay Abstract:
    HALOFUGINONE INHIBITION OF TGF-BETA SIGNALING: TREATMENT FOR BONE METASTASES. BACKGROUND - BONE METASTASIS: One of every eight women will develop breast cancer during her lifetime, and 80% of those women who progress to an advanced stage of disease will develop bone metastases. This devastating complication of cancer causes enormous suffering: bone pain, fractures, hypercalcemia and nerve compression syndromes of paralysis. Bone metastases are incurable, and improved therapies to treat or prevent bone metastases are needed. When tumor cells arrive in bone, they stimulate the cells responsible for bone destruction. The consequent bone destruction not only causes many of the symptoms of metastases but also releases active factors from the bone matrix, in particular transforming growth factor-beta (TGF-beta), which stimulates the tumor cells. Interactions between tumor and bone drive a ?vicious cycle? that perpetuates bone metastases. BACKGROUND - TRANSFORMING GROWTH FACTOR BETA: TGF-beta is stored in bone and drives a vicious cycle of breast cancer bone metastasis. Active TGF-beta is released from bone matrix by cells that resorb bone (osteoclasts) and acts on tumor cells to increase secretion of factors that stimulate further bone destruction. The vicious cycle can be interrupted by inhibitors of TGF-beta signaling. PRELIMINARY DATA: The Principal Investigator?s laboratory and others have shown that inhibition of TGF-beta signaling in breast cancer cells reduces bone metastases, using a dominant-negative TGF-beta receptor, and more recently a small-molecule inhibitor of the type I receptor kinase. However, these approaches have not been carried forward to the clinic. The candidate has shown that halofuginone inhibits TGF-beta signaling in breast cancer cells. PHARMACOLOGY: HALOFUGINONE (Hfg) is a synthetic derivative of a traditional Chinese herbal medicine. Hfg is widely used as a veterinary agent with an excellent safety profile. It increases expression of Smad7, an intracellular inhibitor of TGF-beta signaling. In cancer animal models, it shows anti-angiogenic, anti-metastatic and anti-proliferative effects and it has completed Phase I clinical trials in cancer patients. HYPOTHESIS: HALOFUGINONE WILL EFFECTIVELY INHIBIT BREAST CANCER METASTASES TO BONE IN A PRECLINICAL ANIMAL MODEL SPECIFIC AIM 1: TO TEST THE ABILITY OF HFG TO DECREASE BREAST CANCER BONE METASTASES. Mice receiving breast cancer cells in a model that results in consistent metastases to bone will be treated with drug and changes in tumor burden in bone analyzed. SPECIFIC AIM 2: TO TEST IF THE EFFECTS OF HFG AGAINST TUMOR ARE SPECIFIC TO BONE. Breast cancer cells will be grown as mammary fat pad tumors and the effects of Hfg treatment on tumor burden compared to the effects in bone from Aim 1. SPECIFIC AIM 3: TO DETERMINE IF THE ACTIONS OF HFG ARE THROUGH INHIBITION OF THE TGF-BETA SIGNALING PATHWAY. The effects of Hfg treatment will be compared, as in Aim 1, to those caused by a small molecule inhibitor of the TGF-beta receptor. Results from experiments in vivo will be compared to changes seen in vitro in activity of Smad proteins, which are TGF-beta signaling pathway components. RESEARCH SIGNIFICANCE: Bone metastases are an incurable complication of breast cancer. They occur in the majority of women dying from advanced disease and contribute to substantial morbidity: causing pain, fracture, spinal cord compression, hypercalcemia and cachexia. TGF-beta is an established target for intervention to decrease breast cancer metastases to bone, but practical treatments that could be quickly brought to the clinic are lacking. Hfg offers a novel approach to treating bone metastases by inhibiting TGF-beta. It is orally active, has low toxicity, and has an extensive safety record in animals. Hfg is entering Phase II trials and could rapidly be brought to the clinic for the specific treatment of bone metastases in breast cancer patients. TRAINING SIGNIFICANCE: The mentor, Theresa A Guise, MD, has pioneered the study of TGF-beta in breast cancer bone metastases and trained many active investigators in the field of bone metastases. The environment will train the candidate to sustain a career in preclinical development of agents that target bone metastases in breast cancer.