Abstract 1437: Enhancer of zeste homologue 2 (EZH2) knockdown reveals molecular drivers of EMT and metastasis in triple negative breast cancer (TNBC)

Abstract EZH2 drives TNBC metastasis by promoting epithelial-to-mesenchymal transition (EMT), enhancing cancer stem cell traits, and activating pro-metastatic pathways like Wnt/β-catenin and TGF-β signaling. It also represses tumor suppressor genes through H3K27 trimethylation. EZH2 is overexpressed in TNBC, promoting tumor progression via epigenetic silencing, stemness, EMT, and oncogenic pathways, causing drug resistance and reducing the efficacy of therapy. We hypothesized that silencing EZH2 could uncover its target key genes and pathways responsible for driving metastasis in TNBC. EZH2 knockdown was achieved by CRISPR-cas9 in MDA-MB-231 cells. EZH2 knockdown was confirmed by Western blotting. To test the proper formation of spheroid, we employed a 3D culture system of MDA-MB-231 parental and EZH2 KO cells encapsulated in calcium-alginate microgels seeded from a microfluidic droplet generator. The knockdown of EZH2 inhibited tumor spheroid formation and displayed cytoskeleton de-arrangement. The knockdown of EZH2 also attenuated TNBC growth, migration and proliferation in vitro. RNA from parental MDA-MB-231 cells harboring the cas9 control and the EZH2 knockdown (KO) MDA-MB-231 cells was isolated in quadruplets and used for gene expression profiling using RNA sequencing. Differential gene expression (DGE) analysis was performed using edgeR. KO of EZH2 in TNBC and resulted in the upregulation of 1, 096 genes and the downregulation of 1, 010 genes. Differential gene expression analysis revealed alterations of expression of key regulators of processes such as EMT, migration, and the tumor microenvironment remodeling such as: EGF, IL6, IL6R, JAG1/2, WNT5A, ITGA2/5/V, FN1, and GDF15. Knockdown of EZH2 had a substantial effect on TNBC growth, disrupting the regulation of essential genes and pathways critical for metastasis. Enrichment analysis of the differentially expressed genes identified key biological functions and pathways associated with EZH2 knockdown, including extracellular matrix organization (M610), collagen formation (M631), focal adhesion (MM15913), and degradation of the extracellular matrix (M587). These pathways are crucial for tumor invasion and metastasis, emphasizing the role of EZH2 in regulating the tumor microenvironment. Our findings demonstrate that EZH2 knockdown significantly impairs TNBC growth in vitro and disrupts the regulation of key genes and pathways central to metastasis. (This work is supported by DOD: W81XWH2010065, for Eswar Shankar). Citation Format: Maciej Pietrzak, Xilal P. Rima, Gautam Sarathy, Shivani Dhekne, Lara Rizzotto, Dario Palmieri, Sanjay Gupta, Daniel Stover, Eduardo Reátegui, Christian Rolfo, Eswar Shankar. Enhancer of zeste homologue 2 (EZH2) knockdown reveals molecular drivers of EMT and metastasis in triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1437.