Abstract 29: RANK-RANKL signaling inhibition delays early breast cancer bone metastasis formation
Authors: Sousa Sofia , Gineyts Evelyne , Geraci Sandra , Croset Martine , Clézardin Philippe 
Published: August 2018

Abstract
Denosumab is a fully human monoclonal antibody to human RANKL, currently approved in the management of established bone metastases, mainly due to its anti-resorptive activity which blocks tumor-induced osteoclastogenesis and osteolysis.
Our aims are to determine the role of RANK expression in breast cancer (BC) bone metastasis and the efficacy of RANKL blocking by AMG161 (IgG1 equivalent to Denosumab). AMG161 does not recognize murine RANKL, so we use a BalB/c nude knock-in (KI) mouse model, which expresses a chimeric murine/human RANKL. Moreover, non-invasive in vivo imaging techniques such as bioluminescence and µCT were used to monitor metastatic tumor burden and bone degradation, respectively. To evaluate bone marrow early colonization by BC cells, DiD labelling and ex vivo laser scanning multiphoton confocal microscopy were used.
Long-term studies showed that intra-arterial (i.a.) injection of MDA-MB231 RANK versus MDA-MB231 leads to a poorer overall survival (OS), with the most striking differences seen in the earlier time-points. In this respect, the extent of radiographic osteolytic lesions was higher in the RANK overexpressing group than in the control group. Similar findings were observed following intra-tibial inoculation of RANK-expressing MCF-7 cells, when compared to that observed with parental MCF-7 cells. However, at later time points, bone and lung metastatic burden was similar between the two groups, suggesting that the poor OS of the RANK overexpressing group was due to a faster tumor progression owing to enhanced bone homing and colonization. Indeed, using short-term studies (5 and 14 days), i.a. injection of MDA-MB231 RANK led to higher incidence and extension of bone marrow micro-metastases than MDA-MB231, whereas there was no difference for lung micro-metastases. Daily subcutaneous injections of 1,5mg/kg AMG161 antibody to MDA-MB231RANK tumor-bearing animals, decreased bone micro-metastases and early bone marrow colonization without affecting lung micro-metastasis.
We therefore hypothesized that RANK overexpressing BC cells interact with RANKL produced by osteocytes, osteoblasts and other cells in the osteogenic niche, favoring survival and colonization of BC cells in bone. To test this hypothesis, we made use of heterotypic mammosphere assays in which BC cells were cultured with or without cells of the osteogenic niche (e.g. pre-osteoblasts, mesenchymal stem cells), recombinant RANKL and anti-RANK antibody or AMG161. Co-culture with recombinant RANKL or a cellular source of RANKL increased BC cell mammosphere formation, and blocking RANK/RANKL signaling impaired it.
In conclusion, RANK/RANKL signaling promotes the early engraftment of BC cells in the bone marrow. Blocking this signaling with AMG161 decreases bone marrow micro-metastasis formation in vivo, suggesting that adjuvant treatment of early-stage breast cancer patients with denosumab may prevent bone relapse.
Citation Format: Sofia Sousa, Evelyne Gineyts, Sandra Geraci, Martine Croset, Philippe Clézardin. RANK-RANKL signaling inhibition delays early breast cancer bone metastasis formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 29.