Abstract 3786: Osteocytic connexin 43 hemichannels in the inhibition of cancer cell migration.
Authors: Zhou Jade Z. , Jiang Jean X. 
Published: August 2014

Connexin 43 (Cx43) can form both gap junction channels and halves of gap junctions known as hemichannels. Hemichannels allow communication between cells and the extracellular environment. Cx43 hemichannels in osteocytes have been shown to open when induced by mechanical stimulation through fluid flow shear stress (FFSS) as well as by treatment with alendronate (AD), an efficacious and commonly used bisphosphonate drug. The opening of hemichannels is known to release small molecules important for bone formation and remodeling. Here, we explore the role of Cx43 hemichannels in osteocytes and how it affects cancer cell migratory behavior. We confirm the opening of Cx43 hemichannels in the presence of AD by measuring uptake of ethidium bromide (EtBr) in osteocytic MLO-Y4 cells. As expected, an increase in EtBr uptake was observed after AD treatment. To ensure the specificity of Cx43 hemichannels, we treated the MLO-Y4 cells with Cx43 (E2) antibody, an antibody which specifically blocks Cx43 hemichannel activity. This prevented the uptake of EtBr by the MLO-Y4 cells. To elucidate the effect(s) of osteocyte Cx43 hemichannels on cancer cell migration, we stimulated the opening of Cx43 hemichannels on MLO-Y4 osteocytes with either AD or FFSS. Conditioned media (CM) was collected after treatment and were used to incubate with metastatic MDA-MB-231 breast cancer cells and PC3 prostate cancer cells. Cell migration was measured using wound healing and transwell migration assays. The CM collected from MLO-Y4 osteocytes treated with AD or by FFSS reduced cancer cell migration in a dose-dependent manner. In order to elucidate the direct functional involvement of Cx43 hemichannels in cell migration, we treated the MLO-Y4 cells with Cx43 (E2) antibody. Treatment with this antibody significantly attenuated the inhibitory effect of the CM on the migration of MDA-MB-231 and PC3 cells. Interestingly, the inhibitory effects on cancer cell migration were not observed when we used CM collected from osteoblasts, whose hemichannels were not shown to open by AD treatment. In order to determine whether ATP released from the Cx43 hemichannels in osteocytes would have an effect on cancer cell migration, we depleted ATP from the CM collected from MLO-Y4 cells using apyrase, an ATP hydrolyzing enzyme. The addition of apyrase increased MDA-MB-231 cell migration. To test the effect of purinergic signaling on breast cancer cell migration, we treated the CM with oxidized ATP (oATP), a potent inhibitor of ATP P2X receptors. The addition of oATP attenuated the inhibitory effect of CM on MDA-MB-231 cell migration. Then we stimulated the purinergic receptors with a nonhydrolyzable ATP analogue, ATPγS, the migration of MDA-MB-231 cells decreased. Together, our results point to the role of adenosine nucleotides released from Cx43 hemichannels in tumor migration and metastasis.

Citation Format: Jade Z. Zhou, Jean X. Jiang. Osteocytic connexin 43 hemichannels in the inhibition of cancer cell migration. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3786. doi:10.1158/1538-7445.AM2013-3786