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

Abstract

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.

Tumor-Stroma Crosstalk in Bone Tissue: The Osteoclastogenic Potential of a Breast Cancer Cell Line in a Co-Culture System and the Role of EGFR Inhibition

“…Osteoclasts are not the only TRAP-positive polinucleated cells of a monocytic origin. In a previous work we confirmed that the osteoclast cell-like cells positive to TRAP are real osteoclasts, as we observed the presence of two specific osteoclast markers, i.e., Actin ring and calcitonin receptor (CTR), both in the negative control and in the differentiation medium (DM) condition [ 12 ].…”

“…Human pre-osteoclasts were generated from PBMC, following a previously established protocol with some modifications [ 12 ]. Briefly, PBMC of healthy donors who had given their written informed consent were separated by Ficoll density centrifugation (Lymphosep, Biowest, Nuaillé, France), counted and seeded in αMEM medium (Lonza) supplemented with 10% FBS, 1% penicillin/streptomycin, 1% glutamine (referred to as complete αMEM), in 24-well plates at a density of 750,000 cells/cm 2 .…”

“…Everolimus (Eve) has been recently considered suitable for targeting bone metastases as it inhibits both cancer cells and osteoclasts. [ 12 , 13 , 14 ]. EGFR, also known as ErbB-1 or HER1, is one of the four members of the ErbB tyrosine kinases receptors family.…”

Kaposiform lymphangiomatosis treated with multimodal therapy improves coagulopathy and reduces blood angiopoietin‐2 levels

“…Therefore, additional medical regimens were discussed. Bisphosphonates in combination with sirolimus have suggested a synergistic effect when utilized in complicated lymphatic anomalies of the bone (GLA and Gorham Stout Disease) 11,12 and in the treatment of malignancies with metastatic bone disease 16 . Given his bone involvement and continued thrombocytopenia, around 18 months after diagnosis the patient was initiated on a trial of zoledronate (0.025 mg/kg intravenously monthly for 6 months, then every 3 months for 1 year).…”

Concurrent antitumor and bone-protective effects of everolimus in osteotropic breast cancer

“…Despite these considerations, a recent study supports our observations of everolimus as being an inhibitor of osteoclastogenesis. When investigating the effects of everolimus on the osteoclastogenesis of peripheral blood monocytes in a coculture model with the triple-negative breast cancer cell line SCP2, everolimus was effective at inhibiting osteoclastogenesis induced by SCP2-derived factors [ 37 ].…”