The Effect of Everolimus in an In Vitro Model of Triple Negative Breast Cancer and Osteoclasts

Int J Mol Sci. 2016 Nov 1;17(11):1827. doi: 10.3390/ijms17111827.


Metastatic bone disease has a major impact on morbidity of breast cancer (BC) patients. Alterations in mTOR signaling are involved both in cancer progression and in osteoclast differentiation. The purpose of this study was to assess the role of mTOR inhibitor Everolimus (Eve) on osteoclastogenesis induced by triple negative BC cells. To this aim, we developed an in vitro human model of osteoclastogenesis from peripheral blood monocytes co-cultured with the triple negative SCP2 and the hormonal receptor positive MCF7 cell lines. Osteoclastogenesis was evaluated by TRAP staining, evaluation of F actin rings and Calcitonin Receptor expression. Eve significantly reduced differentiation induced by cancer cells and resulted more effective when evaluated in combination with Denosumab and Zoledronic Acid (Zol). Combination with Zol showed a total abrogation of osteoclast differentiation induced by the triple negative cell line, not by MCF7. Finally, we observed that Eve was active in the inhibition of the crosstalk between cancer cells and osteoclasts reproduced by our model, highlighting a new therapeutic choice for the subsetting of triple negative BC patients. We observed a difference in the response to bone-targeted therapy with respect to BC subtypes. Our model may represent a valid platform for preclinical trials on bone-targeted drugs and for the study of the interplay of BC with bone stromal cells.

Keywords: bone metastasis; breast cancer; everolimus; triple negative.

MeSH terms

  • Antineoplastic Agents / pharmacology
  • Biomarkers / metabolism
  • Blotting, Western
  • Bone Neoplasms / genetics
  • Bone Neoplasms / metabolism
  • Bone Neoplasms / secondary
  • Cell Differentiation / drug effects*
  • Cell Differentiation / genetics
  • Cell Line, Tumor
  • Cells, Cultured
  • Coculture Techniques
  • Denosumab / pharmacology
  • Diphosphonates / pharmacology
  • Everolimus / pharmacology*
  • Gene Expression / genetics
  • Humans
  • Imidazoles / pharmacology
  • Intercellular Adhesion Molecule-1 / metabolism
  • Interleukin-6 / metabolism
  • MCF-7 Cells
  • Models, Biological
  • Osteoclasts / cytology
  • Osteoclasts / metabolism*
  • Osteonectin / genetics
  • RANK Ligand / metabolism
  • Receptors, CXCR4 / genetics
  • Reverse Transcriptase Polymerase Chain Reaction
  • Transforming Growth Factor beta / genetics
  • Triple Negative Breast Neoplasms / genetics
  • Triple Negative Breast Neoplasms / metabolism*
  • Triple Negative Breast Neoplasms / pathology
  • Zoledronic Acid


  • Antineoplastic Agents
  • Biomarkers
  • CXCR4 protein, human
  • Diphosphonates
  • ICAM1 protein, human
  • Imidazoles
  • Interleukin-6
  • Osteonectin
  • RANK Ligand
  • Receptors, CXCR4
  • SPARC protein, human
  • TNFSF11 protein, human
  • Transforming Growth Factor beta
  • Intercellular Adhesion Molecule-1
  • Denosumab
  • Zoledronic Acid
  • Everolimus