Abstract 1011: Prolactin-induced EDD E3 ubiquitin ligase regulates pro/anti-apoptotic gene expression, and stimulates TORC1 signaling and drug resistance in breast cancer cells

Abstract

Abstract EDD E3 ubiquitin ligase or UBR5, first identified as a progestin-inducible gene in human T47D breast cancer cells, mediates DNA damage signal transduction and is a key regulator of various cellular processes in cancer. We identified EDD as a prolactin (PRL)-inducible gene and as a novel partner of the TORC1-associated α4 phosphoprotein-PP2Ac phosphatase complex that regulates the initiation of translation and cell cycle progression. EDD targets PP2Ac for proteasomal degradation. Our aim was to further study the role of EDD in breast cancer cell survival, PRL-stimulated TORC1 signaling and drug resistance. We showed that EDD gene expression varied in breast cancer cell lines of different subtypes, such as luminal-A (MCF7, T47D), HER2-enriched (SkBR3), triple-negative claudin-low (MDA-MB-231) and basal-like (MDA-MB-436, -468). In high EDD-expressing MCF7 and T47D cells, EDD knockdown with siRNA (siEDD) significantly decreased cell viability, increased apoptosis, and arrested cells in the G2-phase of the cell cycle. Specifically, MCF-7 cells in late-stage apoptosis increased significantly (P=0.043, n=5) from a range of 2 - 8% in control cells to 20 - 35 % in siEDD-transfected cells. Although T47D cells were less affected by EDD loss, cells in early-stage apoptosis increased from 4 - 5% in control cells to 13 - 20 % in siEDD-transfected cells. SiEDD-induced apoptosis in MCF7 cells correlated with a significant increase in pro-apoptotic Bim and Bak mRNAs/proteins, leading to increased cleavage of caspase-7 at 6 - 48 h and consequently, cleavage of caspase substrate PARP-1 at 24 - 48 h, as compared to control cells. EDD loss in MCF7 cells decreased PRL-induced phosphorylation of eukaryotic initiation factor 4E-binding protein (4EBP1), a mediator of TORC1 signaling. Specifically, doublet α- and β-bands of 4EBP1 were detected in control cell lysates. A hyper-phosphorylated γ-band appeared in lysates of cells treated with PRL for 1 h but this γ-band disappeared in cells with EDD loss, implicating PRL-induced EDD enhancement of TORC1 signaling. Lastly, in low EDD-expressing MDA-MB-436 cells, EDD gain following pCMV-Tag2.EDD transfection increased cell resistance to chemotherapeutic drugs cisplatin and doxorubicin, and TORC1/TORC2 inhibitors rapamycin and INK128, as compared to controls. In contrast, EDD loss in MCF7 cells increased cell sensitivity to these drugs and to selective estrogen receptor modulator tamoxifen. In summary, PRL-induced EDD promotes breast cancer cell survival, enhances 4EBP1 phosphorylation, and increases drug resistance in vitro. These findings implicate EDD as a potential therapeutic target and support PRL receptor blockade as an additional therapy for breast tumours that express this receptor, including the aggressive HER2+ and triple-negative subtypes. Citation Format: Tyler MacDonald, Lynn N. Thomas, Emily Daze, Penelope J. Barnes, Catherine K. Too. Prolactin-induced EDD E3 ubiquitin ligase regulates pro/anti-apoptotic gene expression, and stimulates TORC1 signaling and drug resistance in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1011.