Abstract P3-13-06: Osteocytic Connexin 43 Hemichannels in Prevention of Bone Metastasis

Abstract

Abstract Bone metastases are major, potentially fatal complications in patients with advanced cancers including breast, prostate and lung cancers. The gap junction-forming protein connexin 43 (Cx43) has been known to inhibit primary tumor cell growth. In addition to gap junctions, Cx43 also forms halves of gap junctions known as hemichannels which allow communication between the intracellular and extracellular environments. Cx43 hemichannels in osteocytes have been shown to open when induced by mechanical stimulation 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. To confirm the opening of Cx43 hemichannels in the presence of AD, we measured uptake of ethidium bromide (EtBr) in MLO-Y4 cells after the addition of AD. As expected, an increase in Cx43 hemichannels EtBr uptake was observed after AD treatment. To elucidate the effect(s) of osteocyte Cx43 hemichannels on cancer cell growth and migration, we treated MLO-Y4 osteocytes with AD. 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, and cell proliferation was measured using the soft agarose anchorage-independent growth assay. The CM collected from MLO-Y4 osteocytes treated with AD reduced cancer cell migration and proliferation in a dose-dependent manner. However, direct treatment with AD had no effect on the cell migration or proliferation of the MDA-MB-231 cells. These data validated that the decrease in migration and proliferation was not a result of direct action by AD on the cancer cells. In order to elucidate the direct functional involvement of Cx43 hemichannels in cell migration, we treated the MLO-Y4 cells with Cx43 (E2) antibody, a specific antibody which blocks hemichannel activity but not gap junction channels formed by Cx43. The inhibitory effect of the CM on the migration and proliferation of MDA-MB-231 and PC3 cells was diminished. Interestingly, the inhibitory effects of osteocytes on cancer cell migration were not observed when using osteoblasts. Together, these results suggest the functional importance of osteocytic Cx43 hemichannels in attenuating cancer cell migration and growth. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-13-06.