Mechano-activation of osteocytic connexin hemichannels prime microenvironment in suppressing breast cancer bone metastasis.

Mechanical stress profoundly influences tumor-bone interaction. However, the underlying mechanism is largely elusive. Here, we reveal that mechanical loading activates osteocyte connexin 43 (Cx43) hemichannels (HCs) and promotes the release of soluble factors, thus establishing a tumor-suppressive bone microenvironment that limits the growth of metastatic breast cancer in bone. Conditioned media from fluid flow shear stress (FFSS)-stimulated osteocyte MLO-Y4 cells significantly inhibited tumor spheroid expansion and migration of both triple-negative and ER-positive breast cancer cells. This inhibitory effect was abolished by blockade of Cx43 HCs. FFSS-activated osteocytes also reduced filopodial extensions and epithelial-mesenchymal transition (EMT) marker expression in cancer cells. Mechanistically, FFSS stimulation induced Cx43 HC-mediated release of prostaglandin E2 (PGE2) and ATP, which exerted opposing effects: PGE2 promoted, while ATP suppressed, breast cancer cells. The relative ATP:PGE2 ratio served as a key determinant of the extent of tumor inhibition. In vivo tibial loading for two weeks prior to tumor inoculation further demonstrated the anti-metastatic function of Cx43 HCs. Mechanical loading suppressed breast cancer proliferation, growth, and osteolytic damage in wild-type (WT) and R76W transgenic mice (defective in gap junctions (GJs) but retaining HCs), but not in Δ130-136 mice (defective in both HCs and GJs). Consistently, Cx43(M1)-mediated HC blockade attenuated the tumor-suppressive effects of mechanical loading. ATP and PGE2 levels were elevated in loaded tibiae of WT and R76W, but not Δ130-136 mice. Collectively, these findings identify Cx43 HC activation by mechanical stress as a key mechanism driving the bone's transition from a metastasis-permissive to a metastasis-resistant microenvironment, mediated by ATP-dominant paracrine signaling from osteocytes that suppresses breast cancer progression.