Abstract P3-03-14: Screening strategies for Notch3-degradation-related E3 ligase in breast cancers

BACKGROUND: The Notch pathway is very conserved and used iteratively during development and regulation of cell fates. Deregulation or loss of Notch signaling is associated with a wide range of human disorders, including breast cancer. It is accepted that ubiquitination events are linked to Notch receptors and their ligands where more than 10 E3-ligase and de-ubiquitination enzymes were implicated in this process. In this study, we attempt to apply a workflow to perform an E3-ligase RNAi screen focusing on the degradation system of Notch signaling pathway in breast cancer cells, hoping to further elaborate the pathogenesis of breast cancer and provide new ideas and schemes for its treatment. MATERIALS AND METHODS: Breast cancer cell line MCF7 was treated with cycloheximide (CHX), an inhibitor of protein biosynthesis, to observe the half-life of Notch3 protein. Then a cell-based screening assay was utilized via a 4 × CSL-luc reporter gene, which reacted to a siRNA library targeting E3 ligases that when silenced inhibit proteasome function in Notch signaling pathway. Moreover, Western Blot and Real-Time PCR were conducted to measure various levels of Notch3 after over-expression of potential E3s. Immunoflourescence was checked for co-colonization of potential E3s and Notch3. RESULTS: Since ubiquitination plays a major role in Notch, we performed a siRNA screen targeting human ubiquitin ligases in MCF7 cells. We quantified the activity of 4 × CSL-luc reporter gene as a read-out for Notch3 contents. From the screening results, we identified the APC/C ubiquitin ligase Cdh1 as a potential player in Notch3 degradation. Over-expressed Cdh1 led to decreased Notch3 levels in post-translational level. Moreover, Cdh1-triggered Notch3 led to a reversed accumulation when inhibited by proteasome inhibitor, MG132. CONCLUSION: Taken together, the 4 × CSL-luc reporter system was effective for detecting the potential E3 ligase involved in the degradation of Notch3 through ubiquitination-proteasome system in breast cancer. Citation Format: Jing Liu, Man-Li Zhang, Jia-Xin Shen, Ya Xu, Ming-Heng Yuan, Guo-Jun Zhang. Screening strategies for Notch3-degradation-related E3 ligase in breast cancers [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-03-14.