title: The differential anti-tumour effects of zoledronic acid in breast cancer - evidence for a role of the activin signaling pathway.
abstract: BACKGROUND
  Neo-adjuvant breast cancer clinical trials of zoledronic acid (ZOL) have shown that patients with oestrogen negative (ER-ve) tumours have improved disease outcomes. We investigated the molecular mechanism behind this differential anti-tumour effect according to ER status, hypothesising it may in part be mediated via the activin signaling pathway.
  
  
  METHODS
  The effects of activin A, its inhibitor follistatin and zoledronic acid on proliferation of breast cancer cells was evaluated using either an MTS proliferation assay or trypan blue. Secretion of activin A and follistatin in conditioned medium (CM) from MDA-MB-231, MDA-MB-436, MCF7 and T47D cell lines were measured using specific ELISAs. The effects of ZOL on phosphorylation domains of Smad2 (pSmad2c + pSmad2L) were evaluated using immunofluorescence. Changes seen in vitro were confirmed in a ZOL treated subcutaneous ER-ve MDA-MB-436 xenograft model.
  
  
  RESULTS
  Activin A inhibits proliferation of both ER-ve and oestrogen positive (ER + ve) breast cancer cells, an effect impaired by follistatin. ZOL significantly inhibits proliferation and the secretion of follistatin from ER-ve cells only, which increases the biological activity of the canonical activin A pathway by significantly increasing intracellular pSmad2c and decreasing nuclear accumulation of pSmad2L. In vivo, ZOL significantly decreases follistatin and pSmad2L expression in ER-ve subcutaneous xenografts compared to saline treated control animals.
  
  
  CONCLUSIONS
  This is the first report showing a differential effect of ZOL, according to ER status, on the activin pathway and its inhibitors in vitro and in vivo. These data suggest a potential molecular mechanism contributing to the differential anti-tumour effects reported from clinical trials and requires further evaluation in clinical samples.
authors.affiliation: Academic Unit of Clinical Oncology, University of Sheffield, Medical School, Sheffield, UK. c.wilson@sheffield.ac.uk.
authors.forename: Caroline
authors.initials: C
authors.lastname: Wilson
authors.affiliation: Academic Department of Oncology, University of Sheffield, Medical School, Sheffield, UK. p.d.ottewell@sheffield.ac.uk.
authors.forename: Penelope
authors.initials: P
authors.lastname: Ottewell
authors.affiliation: Academic Unit of Clinical Oncology, University of Sheffield, Medical School, Sheffield, UK. r.e.coleman@sheffield.ac.uk.
authors.forename: Robert E
authors.initials: RE
authors.lastname: Coleman
authors.affiliation: Academic Unit of Clinical Oncology, University of Sheffield, Medical School, Sheffield, UK. i.holen@sheffield.ac.uk.
authors.forename: Ingunn
authors.initials: I
authors.lastname: Holen
mesh_terms: D028341:Activins / Q000378:metabolism*; D000818:Animals; D000970:Antineoplastic Agents / Q000008:administration & dosage* / Q000494:pharmacology; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004164:Diphosphonates / Q000008:administration & dosage* / Q000494:pharmacology; D005260:Female; D038681:Follistatin / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000008:administration & dosage* / Q000494:pharmacology; D061986:MCF-7 Cells; D051379:Mice; D010766:Phosphorylation; D011960:Receptors, Estrogen / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D051899:Smad2 Protein / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays; D000077211:Zoledronic Acid
chemical_list: D000970:Antineoplastic Agents; D004164:Diphosphonates; D038681:Follistatin; D007093:Imidazoles; D011960:Receptors, Estrogen; C497782:SMAD2 protein, human; D051899:Smad2 Protein; C422405:activin A; D028341:Activins; D000077211:Zoledronic Acid