Abstract LB-A33: Mitogen activated protein kinase (MAPK) signaling in neprilysin-hypermethylated breast cancer
Authors: Reagor Caleb , Chaib Mehdi , Conway Rebecca E. 
Published: August 2018

Abstract
Background: Neprilysin (NEP), or membrane metalloendopeptidase (MME), is a cell-surface enzyme that negatively regulates endothelin receptor signaling and is implicated in breast cancer. Our lab recently reported that NEP hypermethylation is a powerful prognostic indicator in stage I and triple negative breast cancer. Reactivation of NEP in MDA-MB-231 cells abrogated both invasion and mitogen activated protein kinase (MAPK) signaling. In this study, we further investigated the downstream signaling implications of neprilysin methylation in breast cancer.
Methods: Methylation beta values from the Illumina HumanMethylation 450k platform and RPPA protein values were downloaded from the BRCA TCGA database. Values were sorted into methylated and unmethylated using a dichotomizing algorithm. Pathway analysis was performed using the CorePathway Database (Max Plank Institute) and analyzed using over-representation analysis with pathway-based sets using all linked databases with minimum overlap of 2 genes and a probability of 0.01 or less. MTT proliferation assays (ThermoFisher) and in vitro Matrigel invasion assays were performed with 10 μM PD98059 (Sigma). We used an MME expression vector from OriGene (RC223013) to over-express neprilysin in MDA-MB-231 cells.
Results: We initially hypothesized that MAPK signaling would be induced in NEP-methylated breast cancer samples. Analyzing the TCGA database for pMEK levels in NEP-methylated and NEP-unmethylated breast cancer samples revealed a non-significant trend of increased pMEK levels in NEP-methylated samples. To determine if other proteins and pathways might be induced in NEP-methylated breast cancer, we analyzed all RPPA probes with higher expression in NEP-methylated compared to NEP-unmethylated cancers. In breast cancer samples with at least 75% tumor cells (low stromal contribution) and NEP-methylation, both MAPK1 and MAPK14 were up-regulated, as well as several MAPK signaling-associated proteins and a number of other cancer-promoting proteins. To experimentally test whether NEP-methylated cells were sensitive to MAPK inhibition, we conducted proliferation assays with MDA-MB-231 cells (NEP hypermethylated/low expression) and MCF7 cells (NEP unmethylated/moderate expression) in the presence and absence of the MEK inhibitor PD98059. MDA-MB-231 proliferation was significantly more inhibited by MAPK inhibition than MCF7 cells. MDA-MB-231 cell invasion was modestly but significantly inhibited in the presence of PD98059. Transfecting neprilysin in MDA-MB-231 cells more potently inhibited invasion than PD98059, but treating NEP-transfected cell lines with PD98059 did not further decrease invasion. These results are consistent with neprilysin modulating MAPK signaling as well as additional cancer-promoting protein pathways.
Conclusion: We conclude that breast cancers with hypermethylated neprilysin have increased expression of a number of cancer-promoting proteins, including multiple proteins associated with MAPK signaling. Our in vitro data supports our hypothesis that while neprilysin likely functions to negatively regulate breast cancer invasion in part through MAPK signaling, neprilysin may regulate additional pathways in breast cancer invasion. These pathways, including MAPK, may be useful therapeutic targets in NEP-methylated breast cancers.
Citation Format: Caleb Reagor, Mehdi Chaib, Rebecca E. Conway. Mitogen activated protein kinase (MAPK) signaling in neprilysin-hypermethylated breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A33.