title: Everolimus restrains the paracrine pro-osteoclast activity of breast cancer cells.
abstract: BACKGROUND
  Breast cancer (BC) cells secrete soluble factors that accelerate osteoclast (OC) differentiation, leading to the formation of osteolytic bone metastases. In the BOLERO-2 trial, BC patients with bone involvement who received Everolimus had a delayed tumor progression in the skeleton as a result of direct OC suppression through the inhibition of mTOR, in addition to the general suppressor effect on the cancer cells. Here, we explored the effect of Everolimus, as mTOR inhibitor, on the pro-OC paracrine activity of BC cells.
  
  
  METHODS
  Both MDA-MB-231 and MCF-7 BC cell lines were incubated with sub-lethal amounts of Everolimus, and their conditioned supernatants were assessed for their capacity to differentiate OCs from PBMC from healthy donors, as well as to interfere with their bone resorbing activity shown on calcium phosphate slices. We also measured the mRNA levels of major pro-OC factors in Everolimus-treated BC cells and their secreted levels by ELISA, and evaluated by immunoblotting the phosphorylation of transcription factors enrolled by pathways cooperating with the mTOR inhibition. Finally, the in vivo pro-OC activity of these cells was assessed in SCID mice after intra-tibial injections.
  
  
  RESULTS
  We found that Everolimus significantly inhibited the differentiation of OCs and their in vitro bone-resorbing activity, and also found decreases of both mRNA and secreted pro-OC factors such as M-CSF, IL-6, and IL-1β, whose lower ELISA levels paralleled the defective phosphorylation of NFkB pathway effectors. Moreover, when intra-tibially injected in SCID mice, Everolimus-treated BC cells produced smaller bone metastases than the untreated cells.
  
  
  CONCLUSIONS
  mTOR inhibition in BC cells leads to a suppression of their paracrine pro-OC activity by interfering with the NFkB pathway; this effect may also account for the delayed progression of bone metastatic disease observed in the BOLERO-2 trial.
authors.affiliation: Department of Biomedical Sciences and Human Oncology, University of Bari "A. Moro", P.zza Giulio Cesare, 11-70124, Bari, Italy. valeriasimo@gmail.com.
authors.forename: Valeria
authors.initials: V
authors.lastname: Simone
authors.affiliation: Department of Biomedical Sciences and Human Oncology, University of Bari "A. Moro", P.zza Giulio Cesare, 11-70124, Bari, Italy. sabinociavarella@yahoo.it.
authors.forename: Sabino
authors.initials: S
authors.lastname: Ciavarella
authors.affiliation: Department of Biomedical Sciences and Human Oncology, University of Bari "A. Moro", P.zza Giulio Cesare, 11-70124, Bari, Italy. dr.oronzo.brunetti@tiscali.it.
authors.forename: Oronzo
authors.initials: O
authors.lastname: Brunetti
authors.affiliation: Department of Biomedical Sciences and Human Oncology, University of Bari "A. Moro", P.zza Giulio Cesare, 11-70124, Bari, Italy. annalisa.savonarola@gmail.com.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Savonarola
authors.affiliation: Department of Biomedical Sciences and Human Oncology, University of Bari "A. Moro", P.zza Giulio Cesare, 11-70124, Bari, Italy. mauro.civ@tiscali.it.
authors.forename: Mauro
authors.initials: M
authors.lastname: Cives
authors.affiliation: Department of Biomedical Sciences and Human Oncology, University of Bari "A. Moro", P.zza Giulio Cesare, 11-70124, Bari, Italy. marco.tucci@uniba.it.
authors.forename: Marco
authors.initials: M
authors.lastname: Tucci
authors.affiliation: Department of Pathological Anatomy, University of Bari "A. Moro", Bari, Italy. giusyopinto@hotmail.it.
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Opinto
authors.affiliation: Department of Pathological Anatomy, University of Bari "A. Moro", Bari, Italy. eugenio.maiorano@uniba.it.
authors.forename: Eugenio
authors.initials: E
authors.lastname: Maiorano
authors.affiliation: Department of Biomedical Sciences and Human Oncology, University of Bari "A. Moro", P.zza Giulio Cesare, 11-70124, Bari, Italy. francesco.silvestris@uniba.it.
authors.forename: Franco
authors.initials: F
authors.lastname: Silvestris
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001859:Bone Neoplasms / Q000378:metabolism / Q000556:secondary; D001862:Bone Resorption / Q000188:drug therapy / Q000235:genetics; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D002454:Cell Differentiation / Q000187:drug effects; D045744:Cell Line, Tumor; D016207:Cytokines / Q000096:biosynthesis; D004195:Disease Models, Animal; D000068338:Everolimus / Q000494:pharmacology*; D005260:Female; D020869:Gene Expression Profiling; D064593:Heterografts; D006801:Humans; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D019899:Paracrine Communication / Q000187:drug effects*; D015398:Signal Transduction / Q000187:drug effects; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D016207:Cytokines; D016328:NF-kappa B; D000068338:Everolimus; D058570:TOR Serine-Threonine Kinases