Abstract 1556: Tumor-associated osteoblasts are major mediators in facilitating bone metastatic breast cancer cell quiescence

Abstract

Abstract Breast cancer (BC) has a predilection for bone metastasis, where the five-year survival rate is bleak (<10%). Deadly disseminated BC cells invade the bone and can remain undetectable and untreatable for ≤25 years during a period of proliferative quiescence. We have evidence that disseminated BC cells create a proliferatively quiescent niche by co-opting osteoblasts (OBs) to alter their production of microRNAs that regulate cell cycle and cellular dormancy. MC3T3-E1 murine OBs were grown to various stages of maturity: 4 (growth), 10 (early differentiation), and 20 days (late differentiation) and incubated with conditioned medium (CM) from human BC MDA-MB-231 cell variants (parental MDA-231 or metastasis-suppressed MDA-231BRMS) to produce tumor-associated osteoblasts (TAOs). TAO-derived exosomes were assayed for microRNAs (miRs) using species-specific Qiagen miScript miRNA PCR arrays. Also, tumors from mice inoculated via flank injection with TAO cells plus MDA-231-GFP BC cells were assayed ex-vivo for RUNX2 production. Metastasis-suppressed BC cells formed Connexin 43 gap junctions and exchanged cellular material with TAOs as determined by a parachute assay. Additionally, TAO cells produced substantial amounts of exosomes, with the largest induction seen in TAOs generated from metastasis-suppressed BC CM, that were taken up by the BC cells. TAO-derived exosomes were found to contain increased amounts of microRNAs that decrease cellular proliferation and induce G0 phase of the cell cycle (miR 320a), and repress cellular proliferation and regulate cyclin D1 expression (miR 193b). Knock-down of miRs 320a and 193b in BC cells resulted in a reduced number of cells in G0 and increased numbers of cells in G1/S/G2/M phases of the cell cycle as indicated by acridine orange and propidium iodide staining. This same phenomenon was found in-vivo in mice inoculated with TAO cells plus proliferatively quiescent BC cells. Tumors composed of an admix of TAO cells plus BC cells grew slower and were not as large as tumors composed of OBs plus BC cells, or BC cells alone. Furthermore, after over 60 days in-vivo, tumors composed of TAO cells plus BC cells exhibited the presence of OB differentiation marker RUNX2, indicating that TAOs were still present in the tumor population, contributing to the tumor microenvironment through culmination of the experiment. Combined, these data suggest that TAOs produce exosomal miRs that contribute to the induction of BC cell proliferative quiescence in the bone. The nature of this study also suggests the importance of OBs and their crosstalk in orchestrating the proliferative quiescence of disseminated BC cells in the bone. Thus, these findings clearly implicate the bone microenvironment and cancer cell manipulation thereof in facilitating disseminated tumor cell colonization and survival. Supported by NIH 1RC1 CA146381, 1R01NS06994, P50 CA083639 for FCM; (NRSA) T32 CA079448, NIH 2 K99 CA178177 for KMB. Citation Format: Karen M. Bussard, Frank C. Marini. Tumor-associated osteoblasts are major mediators in facilitating bone metastatic breast cancer cell quiescence. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1556.