ARTICLE ABSTRACTPI3K/AKT/mTOR signaling plays an important role in breast cancer. Its interaction with estrogen receptor (ER) signaling becomes more complex and interdependent with acquired endocrine resistance. Targeting mTOR combined with endocrine therapy has shown clinical utility; however, a negative feedback loop exists downstream of PI3K/AKT/mTOR. Direct blockade of AKT together with endocrine therapy may improve breast cancer treatment. AZD5363, a novel pan-AKT kinase catalytic inhibitor, was examined in a panel of ER+ breast cancer cell lines (MCF7, HCC1428, T47D, ZR75.1) adapted to long-term estrogen deprivation (LTED) or tamoxifen (TamR). AZD5363 caused a dose-dependent decrease in proliferation in all cell lines tested (GI50 < 500 nmol/L) except HCC1428 and HCC1428-LTED. T47D-LTED and ZR75-LTED were the most sensitive of the lines (GI50 ∼100 nmol/L). AZD5363 resensitized TamR cells to tamoxifen and acted synergistically with fulvestrant. AZD5363 decreased p-AKT/mTOR targets leading to a reduction in ERα-mediated transcription in a context-specific manner and concomitant decrease in recruitment of ER and CREB-binding protein (CBP) to estrogen response elements located on the TFF1, PGR, and GREB1 promoters. Furthermore, AZD5363 reduced expression of cell-cycle–regulatory proteins. Global gene expression highlighted ERBB2-ERBB3, ERK5, and IGFI signaling pathways driven by MYC as potential feedback-loops. Combined treatment with AZD5363 and fulvestrant showed synergy in an ER+ patient-derived xenograft and delayed tumor progression after cessation of therapy. These data support the combination of AZD5363 with fulvestrant as a potential therapy for breast cancer that is sensitive or resistant to E-deprivation or tamoxifen and that activated AKT is a determinant of response, supporting the need for clinical evaluation. Mol Cancer Ther; 14(9); 2035–48. ©2015 AACR.