Neprilysin: A potential regulator of PI3K/AKT signaling in triple negative breast cancer cells

Triple-negative breast cancers (TNBC) are among the most aggressive and therapeutically resistant breast cancers and comprise approximately 15-20% of all breast cancer cases. The lack of hormone receptor and HER2 expression makes these cancers inherently resistant to therapies targeting these receptors, yet many TNBC are also resistant to traditional chemotherapies. Thus, identifying molecular targets and predicting sensitivity to these targets is essential for the successful treatment of patients with TNBC. The phosphoinositide 3-kinase (PI3K) pathway is the most commonly mutated pathway in TNBC, suggesting that its inhibition could be a successful therapeutic approach. However, few clinical trials testing PI3K inhibitors in these breast cancers have shown sustained responses. Recent studies classifying TNBC into additional molecular subtypes have identified specific subtypes of TNBC that may be more sensitive to PI3K-targeted therapy, yet early clinical trials reveal heterogeneous patient responses within these subtypes, emphasizing the need to more completely understand the molecular mechanisms driving TNBC to better predict patient response to targeted therapies. Recent studies in our lab implicate neprilysin, a cell-surface protease expressed on breast epithelial and stromal cells, as a potential regulator of PI3K in TNBC. We previously reported that neprilysin negatively regulates breast cancer invasion and is silenced through promoter methylation in some aggressive breast cancers. The purpose of the current study was to determine whether neprilysin is associated with cancer-related proteins in triple negative breast cancer cells. We initially conducted a bioinformatics screen of 15 TNBC cell lines to compare reverse phase protein array (RPPA) protein expression in cell lines expressing high and low levels of neprilysin mRNA. This analysis revealed 13/26 (50%) significantly differentially expressed proteins that are directly involved in PI3K signaling, suggesting a link between neprilysin and PI3K signaling. To further assess this association, we transfected a neprilysin expression vector in MDA-MB-231 triple negative breast cancer cell lines and measured phosphorylation of all AKT isoforms at T308 and of AKT1 and AKT2 at S473 using Western blots. Our results demonstrate that in MDA-MB-231 cells, neprilysin expression decreases AKT phosphorylation at T308 and decreases AKT1 phosphorylation at S473; no significant differences in AKT2 phosphorylation were observed. Together, our results suggest that neprilysin may be negatively regulating PI3K/AKT signaling, which could have important implications for sensitivity of TNBC with PI3K-pathway mutations and high neprilysin expression to PI3K-targeted therapies. Citation Format: Rebecca E. Conway, Katherine Iglesias, Zuhaila Hired, Grace Rutledge, Khadija Kirmani. Neprilysin: A potential regulator of PI3K/AKT signaling in triple negative 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 1785.