Abstract EBP1, an ErbB3 binding protein isolated in our laboratory, sensitizes breast cancer cells to tamoxifen in part by decreasing ErbB2 protein levels. The p21 regulated serine/threonine kinase PAK1, implicated in tamoxifen resistance, phosphorylates EBP1 in vitro and in vivo at T261. Phosphorylation of EBP1 at this site induces tamoxifen resistance. We thus postulated that inhibition of PAK1 activity, by restoring EBP1 function, could ameliorate the hormone refractory phenotype of ErbB2 overexpressing breast cancer cells. Transient transfection studies indicated that an EBP1 T261E mutant, which mimics EPB1 phosphorylated by PAK1, increased ErbB2 protein levels. An EBP1 T261A mutant, unable to be phosphorylated by PAK1, ameliorated PAK1-induced tamoxifen resistance, suggesting that phosphorylation of EBP1 by PAK1 contributes to tamoxifen resistance. We then tested if pharmacological inhibition of PAK1 activity might render hormone resistant cells, which endogenously overexpress PAK1 and ErbB2, tamoxifen sensitive. Transfection of EBP1 sensitized cells to tamoxifen. The T261A EBP1 mutant more potently sensitized cells to tamoxifen than wild type EBP1. IPA-3, a specific small MW PAK1 inhibitor, sensitized cells to tamoxifen only when EBP1 was ectopically expressed. However, IPA-3 did not further affect sensitivity of T261A overexperssing cells, suggesting that the phosphorylation of EBP1 by PAK1 played a role in the EBP1-induced hormone sensitivity. IPA had no effect on tamoxifen resistance in T47D cells in which EBP1 protein had been ablated by shRNA. The IPA-induced increase in tamoxifen sensitivity was accompanied by a decrease in ErbB2 levels in EBP1 overexpressing, but not vector control cells. These studies suggest that phosphorylation of EBP1 may be a novel additional mechanism of PAK1-induced hormone resistance. In addition, strategies that increase the pool of functional EBP1 and decrease PAK1 activity may affect hormone resistant cell growth. Our results also suggest that PAK1 directed therapies may be more effective in patients that express high levels of EBP1. *contributed equally to this work Citation Format: Smita Awasthi*, Arundhati Ghosh*, Jeffrey R. Peterson, Anne W. Hamburger. Regulation of tamoxifen sensitivity by a PAK1-EBP1 signaling pathway in breast cancer. [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 4397. doi:10.1158/1538-7445.AM2013-4397