Abstract 1560: Breast tumor disrupt gap junction mediated vessel stabilization by two distinct mechanisms

On normal blood vessels, vascular mural cells (pericytes and smooth muscle cells) serve to stabilize the vessel and render the endothelium quiescent. Tumor vessels, in contrast, exhibit abnormal association between mural cells and endothelial cells which allows the endothelium to be angiogenic. The mechanism(s) by which tumors prevent mural-cell mediated vessel stabilization have not been well elucidated. We have previously demonstrated that heterotypic connexin 43 (Cx43)-dependent gap junction activity is required for mural cell-induced quiescence of endothelial cells, and that long term exposure of mural cells to media conditioned by aggressive MDA-MB-231 breast tumor cells downregulates Cx43 mRNA and protein to release endothelial cells from mural cell-mediated growth inhibition. Since Cx43 function and half-life can also be modulated by phosphorylation, we investigated the role of phosphorylation in tumor-induced regulation of Cx43 function. In the present study, we show that short-term exposure of mural cells to tumor conditioned media (CM) activates PKC signaling in mural cells, resulting in inactivating phosphorylation of Cx43 at the PKC site S368 that is both rapid and sustained. This tumor-induced inactivating S368 phosphorylation occurs in both fully differentiated mural cells (vascular smooth muscle cells [vSMC] and microvascular pericytes) as well as C3H10T1/2 mural precursor cells. Although tumor CM activates several of the PKC isoforms expressed by vSMC, using a panel of pharmacological inhibitors, we have identified PKCΔ as the isoform responsible for inhibitory phosphorylation at S368. We confirmed that the mechanisms by which tumors inactivate Cx43, by mRNA downregulation and by phosphorylation are distinct, as PKC inhibition prevents S368 phosphorylation but does not protect against Cx43 loss following long-term tumor CM treatment. Similarly, MDA-MB-231 conditioned media induces inactivating phosphorylation at Cx43 S255; while MEK1/2 inhibitor UO126 prevents S255 phosphorylation, it does not prevent Cx43 protein decrease. Importantly, using the GAP26 peptide we demonstrate that inhibition of gap junction function without protein loss is sufficient to prevent mural cell-mediated endothelial growth inhibition. Finally, we show that the vessels of xenograft tumors undergo the inactivating phosphorylation at Cx43 S368 as well. These data, together with our previous data, suggest that tumors can override mural cell inhibition of endothelial proliferation to promote angiogenesis in two distinct ways: 1) rapid inactivation of intact Cx43 protein by phosphorylation, and 2) long-term mRNA-mediated loss of Cx43 protein. Strategies to prevent vascular Cx43 loss or inactivation may therefore provide a novel means to inhibit angiogenesis or stabilize tumor vasculature for therapeutic gain. (Supported by NIH CA138727) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1560. doi:10.1158/1538-7445.AM2011-1560