Overexpression of a functional calcium-sensing receptor dramatically increases osteolytic potential of MDA-MB-231 cells in a mouse model of bone metastasis through epiregulin-mediated osteoprotegerin downregulation

// Cedric Boudot 1, * , Lucie Henaut 1, * , Ursula Thiem 1 , Sandra Geraci 2 , Mariangela Galante 1 , Paulo Saldanha 1 , Zuzana Saidak 1 , Isabelle Six 1 , Philippe Clezardin 2 , Said Kamel 1 and Romuald Mentaverri 1 1 Inserm U1088, Centre Universitaire de Recherche en Sante, Universite de Picardie Jules Verne, Amiens, France 2 Inserm UMR 1033, LYOS, Lyon, France * These authors have contributed equally to this work Correspondence to: Said Kamel, email: said.kamel@u-picardie.fr Keywords: bone metastasis, breast cancer, calcium-sensing receptor, epiregulin, osteolysis Received: July 15, 2016 Accepted: March 14, 2017 Published: April 10, 2017 ABSTRACT Introduction and Aims: Osteolytic bone metastases are observed in advanced cases of breast cancer. In vitro data suggest that the activity of the calcium-sensing receptor (CaSR) expressed by metastatic cells could potentiate their osteolytic potential. This study aimed to demonstrate in vivo the involvement of the CaSR in breast cancer cells osteolytic potential and to identify potential targets linked to CaSR activity. Methods and Results: MDA-MB-231 stably transfected with plasmids containing either a full-length wild-type CaSR (CaSR-WT), or a functionally inactive dominant negative mutant (CaSR-DN) or an empty vector (EV) were intratibially injected into Balb/c-Nude mice. X-ray analysis performed 19 days after injection showed a dramatic increase of osteolytic lesions in mice injected with CaSR-WT-transfected cells as compared to mice injected with EV- or CaSR-DN-transfected cells. This was associated with decreased BV/TV ratio and increased tumor burden. Epiregulin, an EGF-like ligand, was identified by a DNA microarray as a possible candidate involved in CaSR-mediated osteolysis. Indeed, in vitro , CaSR overexpression increased both epiregulin expression and secretion as compared to EV- or CaSR-DN-transfected cells. Increased epiregulin expression was also detected in osteolytic bone lesions from mice injected with CaSR-WT-transfected MDA-MB-231. In vitro , exposure of osteoblastic cells (HOB and SaOS2) to exogenous epiregulin significantly decreased OPG mRNA expression. Exposure of osteoblastic cells to conditioned media prepared from CaSR-WT-transfected cells also decreased OPG expression. This effect was partially blocked after addition of an anti-epiregulin antibody. Conclusions: Overexpression of a functional CaSR in metastatic breast cancer cells dramatically amplifies their osteolytic potential through epiregulin-mediated OPG downregulation.