Abstract 1480: ZEB1 dimerization is regulated by acetylation and promotes protein stability, interactions, EMT, and NSCLC metastasis

Abstract

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide due to the ability of cancer cells to metastasize. Therefore, it is essential to expand our current knowledge of the biological processes that contribute to metastasis to guide the discovery of novel therapeutic modalities. The Epithelial-to-mesenchymal transition (EMT) is a mechanism for the acquisition of malignant cells, which causes epithelial cells with specialized cell-cell contacts to gain mesenchymal migratory capacity. High expression of the Zinc finger E-box binding homeobox 1 (ZEB1) transcription factor is correlated with poor clinical outcomes of cancer, including therapeutic resistance and EMT-mediated metastasis. ZEB1 has a calculated molecular weight of 125kDa; however, numerous groups have reported discrepancies in the observed molecular weight (approximately 190-250kDa). This has been attributed to dimerization mediated by post-translational modifications (PTMs). Therefore, we performed mass spectrometry and identified a novel PTM - K811 acetylation - that may regulate ZEB1 dimerization and function. To define the role of ZEB1 acetylation, we generated ZEB1 acetyl mimetic (K811Q) and deficient (K811R) mutant-expressing NSCLC cell lines. We hypothesize that ZEB1 dimerization is regulated by K811 acetylation to promote protein stability and lung adenocarcinoma metastasis. We determined that the K811R ZEB1 (125 kDa) has a shorter half-life wild-type and high ubiquitination rate than (WT) ZEB1 and K811Q ZEB1 (~225 kDa), suggesting that disruption of ZEB1 acetylation hinders its dimerization and protein stability. Further, ZEB1 dimer recruits the nucleosome remodeling and deacetylase (NuRD) complex to bind the promoter of its target genes mir200c-141 and SEMA3F. RNA-sequencing revealed that unlike K811R ZEB1, WT ZEB1 and K811Q ZEB1 downregulate the expression of gene sets involved in tight junction, apical junction, and cell-cell junction formation. We also identified six novel ZEB1 acetylation-dependent gene candidates that function in signaling pathways and epithelial cell maintenance; CBLN3, STAP2, PLEKHG6, LAD1, RNF43, and LLGL2. In vitro 2D migration Boyden chamber, 3D invasion assays, and in vivo studies demonstrated that ZEB1 acetylation promotes the invasive and metastatic potential of the NSCLC cell line. Our findings suggest that K811 acetylation regulates ZEB1 dimerization and stability to promote NuRD interaction and lung adenocarcinoma via EMT. Future studies are focused on assessing changes in gene expression programs and chromatin accessibility that are ZEB1 dimerization dependent and that could contribute to NSCLC metastasis. Citation Format: Mabel Giselle Perez-Oquendo, Roxsan Manshouri, Yanhua Tian, Jared Fradette, Don Gibbons. ZEB1 dimerization is regulated by acetylation and promotes protein stability, interactions, EMT, and NSCLC metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1480.