Abstract
Advanced breast cancer predominantly metastasizes to the skeleton, at which point patient prognosis significantly declines concomitant with bone loss, pain, and heightened fracture risk. Given the skeleton's sensitivity to mechanical signals, increased mechanical loading is well-documented to increase bone mass, and it also inhibited bone metastatic tumor formation and progression in vivo, though the underlying mechanisms remain under investigation. Here, we focus on the role of the osteocyte because it is the primary skeletal mechanosensor and in turn directs the remodeling balance between formation and resoprtion. In particular, osteocytic dendrites are important for mechanosensing, but how this function is altered during bone metastatic breast cancer is unknown. To examine how breast cancer cells modulate dendrite formation and function, we exposed osteocytes (MLO-Y4) to medium conditioned by breast cancer cells (MDA-MB231) and to applied fluid flow (2 h per day for 3 days, shear stress 1.1 Pa). When loading was applied to MLOs, dendrite formation increased despite the presence of tumor-derived factors while overall MLO cell number was reduced. We then exposed MLOs to fluid flow as well as media conditioned by MDAs that had been similarly loaded. When nonloaded MLOs were treated with conditioned media from loaded MDAs, their dendrite formation increased in a manner similar to that observed due to loading alone. When MLOs simultaneously underwent loading and treatment with loaded conditioned media, dendrite formation was greatest. To understand potential molecular mechanisms, we then investigated expression of genes related to osteocyte maturation and dendrite formation (E11) and remodeling (RANKL, OPG) as well as osteocyte apoptosis. E11 expression increased with loading, consistent with increased dendrite formation. Though loaded conditioned media decreased MLO cell number, apoptosis was not detected via TUNEL staining, suggesting an inhibition of growth instead. OPG expression was inhibited while RANKL expression was unaffected, leading to an overall increase in the RANKL/OPG ratio with conditioned media from loaded breast cancer cells. Taken together, our results suggest that skeletal mechanical loading stimulates breast cancer cells to alter osteocyte mechanosensing by increasing dendrite formation and downstream resorption.