title: Tumor-expressed adrenomedullin accelerates breast cancer bone metastasis.
abstract: BACKGROUND
  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.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia, PO Box 800733, Charlottesville, VA, 22908, USA. vsiclari@gmail.com.
authors.forename: Valerie A
authors.initials: VA
authors.lastname: Siclari
authors.affiliation: Division of Endocrinology and Metabolism, Department of Medicine, 450 Ray C Hunt Dr, University of Virginia, PO Box 801406, Charlottesville, VA, 22908, USA. kmohamma@iu.edu.|Division of Endocrinology, Department of Medicine, Indiana University School of Medicine, 980 Walnut, St, C321-C, Indianapolis, IN, 46202, USA. kmohamma@iu.edu.
authors.forename: Khalid S
authors.initials: KS
authors.lastname: Mohammad
authors.affiliation: Division of Endocrinology, Department of Medicine, Indiana University School of Medicine, 980 Walnut, St, C321-C, Indianapolis, IN, 46202, USA. drtompki@indiana.edu.|Richard L. Roudebush VA Medical Center, 1481 W 10th St, Indianapolis, IN, 46202, USA. drtompki@indiana.edu.
authors.forename: Douglas R
authors.initials: DR
authors.lastname: Tompkins
authors.affiliation: Division of Endocrinology and Metabolism, Department of Medicine, 450 Ray C Hunt Dr, University of Virginia, PO Box 801406, Charlottesville, VA, 22908, USA. haw5d@virginia.edu.
authors.forename: Holly
authors.initials: H
authors.lastname: Davis
authors.affiliation: Division of Endocrinology and Metabolism, Department of Medicine, 450 Ray C Hunt Dr, University of Virginia, PO Box 801406, Charlottesville, VA, 22908, USA. smokeynsa@aol.com.
authors.forename: C Ryan
authors.initials: CR
authors.lastname: McKenna
authors.affiliation: Division of Endocrinology and Metabolism, Department of Medicine, 450 Ray C Hunt Dr, University of Virginia, PO Box 801406, Charlottesville, VA, 22908, USA. xianghongpeng622@gmail.com.|Division of Endocrinology, Department of Medicine, Indiana University School of Medicine, 980 Walnut, St, C321-C, Indianapolis, IN, 46202, USA. xianghongpeng622@gmail.com.
authors.forename: Xianghong
authors.initials: X
authors.lastname: Peng
authors.affiliation: Division of Endocrinology and Metabolism, Department of Medicine, 450 Ray C Hunt Dr, University of Virginia, PO Box 801406, Charlottesville, VA, 22908, USA. llwessner@gmail.com.
authors.forename: Lisa L
authors.initials: LL
authors.lastname: Wessner
authors.affiliation: Division of Endocrinology and Metabolism, Department of Medicine, 450 Ray C Hunt Dr, University of Virginia, PO Box 801406, Charlottesville, VA, 22908, USA. mniewoln@iu.edu.|Division of Endocrinology, Department of Medicine, Indiana University School of Medicine, 980 Walnut, St, C321-C, Indianapolis, IN, 46202, USA. mniewoln@iu.edu.
authors.forename: Maria
authors.initials: M
authors.lastname: Niewolna
authors.affiliation: Division of Endocrinology and Metabolism, Department of Medicine, 450 Ray C Hunt Dr, University of Virginia, PO Box 801406, Charlottesville, VA, 22908, USA. tguise@iu.edu.|Division of Endocrinology, Department of Medicine, Indiana University School of Medicine, 980 Walnut, St, C321-C, Indianapolis, IN, 46202, USA. tguise@iu.edu.
authors.forename: Theresa A
authors.initials: TA
authors.lastname: Guise
authors.affiliation: Richard L. Roudebush VA Medical Center, 1481 W 10th St, Indianapolis, IN, 46202, USA. asuvanna@iu.edu.|Division of Hematology/Oncology, Department of Medicine, 980 Walnut St, C321-H, Indiana University School of Medicine, Indianapolis, IN, 46202, USA. asuvanna@iu.edu.
authors.forename: Attaya
authors.initials: A
authors.lastname: Suvannasankha
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia, PO Box 800733, Charlottesville, VA, 22908, USA. jmchirgw@iu.edu.|Division of Endocrinology and Metabolism, Department of Medicine, 450 Ray C Hunt Dr, University of Virginia, PO Box 801406, Charlottesville, VA, 22908, USA. jmchirgw@iu.edu.|Division of Endocrinology, Department of Medicine, Indiana University School of Medicine, 980 Walnut, St, C321-C, Indianapolis, IN, 46202, USA. jmchirgw@iu.edu.|Richard L. Roudebush VA Medical Center, 1481 W 10th St, Indianapolis, IN, 46202, USA. jmchirgw@iu.edu.
authors.forename: John M
authors.initials: JM
authors.lastname: Chirgwin
mesh_terms: D000230:Adenocarcinoma / Q000473:pathology / Q000556:secondary*; D053607:Adrenomedullin / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001859:Bone Neoplasms / Q000473:pathology / Q000556:secondary*; D001842:Bone and Bones / Q000378:metabolism* / Q000473:pathology; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D018450:Disease Progression; D005260:Female; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D012333:RNA, Messenger / Q000378:metabolism*
chemical_list: D012333:RNA, Messenger; D053607:Adrenomedullin