Abstract P5-05-15: Activation of osteocytes connexin43 hemichannels suppresses breast cancer bone metastasis, a potential therapeutic target

Abstract

Abstract Bone is a fertile place for metastasized breast cancer cell (BCC), however, mechanical loading is reported to inhibit BCC growth. Osteocytes, representing over 95% of bone cells, are mechanosensitive; they coordinate bone remodeling and modulate the extracellular environment. Mechanical loading activates osteocityc connexin 43 (Cx43) hemichannels, which mediates communication between the intracellular and extracellular space with message of molecules below 1 kDa. We have shown that ATP released by osteocytic hemichannels through the activation of BCC P2X7 receptor, inhibits BCC growth and migration. Here, we evaluate if activation of Cx43 hemichannels in osteocytes plays a critical role in inhibiting breast cancer bone metastasis. Our lab develops a monoclonal antibody, MHC2, which binds to extracellular domain of Cx43 and increases the activity of osteocytic hemichannels. This antibody was immune-reactive in HeLa cells transfected with Cx43 and osteocytes cell line MLO-Y4. It also labeled mouse osteocytes in situ. MHC2 antibody enhanced the activity of Cx43 hemichannels in cells and in vivo osteocytes and promoted the ATP release from osteocytic MLO-Y4 cells. Moreover, conditioned media collected from MHC2-treated osteocytes reduced BCC migration in vitro. The weekly injection of MHC2 reduced the breast cancer growth in bone in WT mice, but not in specific osteocyte Cx43 knockout mice. MHC2 antibody increase levels of lymphocytes T cytotoxic (CD3+/CD8+) and helper (CD3+/CD4+) in tumor and draining lymph nodes. Moreover, the weekly injection of MHC2, in intracardiac injected tumor cells model, in both immunocompetent and immune-compromised mice increased survival rate and reduced tumor spreading. As is known, osteocytic Cx43 hemichannels are permeable to ATP. We showed that P2X7 specific agonist benzoyl ATP reduced in vitro breast cancer cell migration, increased cytoplasmic free Ca2+ and induced formation of tubulin bundles, an indicator of cytoskeleton reorganization. The effect of benzoyl ATP required activation of CamKII in breast cancer cells. Our results suggest that Cx43 hemichannels are likely a potential therapeutic target in treating breast cancer growth and metastasizing to the bone, probably through the enhancement of the extracellular ATP level and the activation of P2X7/CamKII axis. Citation Format: Manuel A. Riquelme, Sumin Gu, Jeffery B. Chavez, Zhiqiang An, Jean X. Jiang. Activation of osteocytes connexin43 hemichannels suppresses breast cancer bone metastasis, a potential therapeutic target [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-05-15.