Abstract 676: ZEB1 acetylation regulates dimerization and protein stability to promote lung adenocarcinoma progression and metastasis
Authors: Perez-Oquendo Mabel G. , Manshouri Roxsan , Gibbons Don 
Published: June 2022

Lung cancer 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 metastasis, which changes polarized epithelial cells into invasive mesenchymal cells. High expression of the Zinc finger E-box binding homeobox 1 (ZEB1) transcription factor is correlated to poor outcomes in cancer, including therapeutic resistance and EMT-mediated metastasis. ZEB1 has a predicted molecular weight of 125kDa; however, multiple 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) mutants in a panel of lung adenocarcinoma cell lines. We hypothesize that ZEB1 acetylation regulates dimerization and protein stability to promote lung adenocarcinoma progression and metastasis. We determined that the acetyl-deficient mutant (125kDa) exhibits a decreased half-life compared to WT and acetylated ZEB1, suggesting that disruption of acetylation hinders dimerization and protein stability. However, the acetyl-mimetic mutant (250kDa) protects ZEB1 from proteasomal degradation brought by the action of the E3 ubiquitin ligase SIAH1. siRNA-mediated silencing of SIAH1 increased the mRNA and protein levels of ZEB1 WT and both acetylation mutants. Importantly, silencing of SIAH1 markedly increased the half-life of 250kDa ZEB1 WT from 18.0hrs to 25.4hrs and acetyl-mimetic from 12.3hrs to 24.0hrs, as well as 125kDa ZEB1 acetyl-deficient from 9.7hrs to 18.3hrs. Although we determined that 250kDa ZEB1 is significantly more stable than 125kDa ZEB1, the increase in the stability by silencing SIAH1 contributes to ZEB1 acetyl-deficient binding at the promoter of its target genes mir200c-141 and SEMA3F. 250kDa ZEB1 WT and acetyl-mimetic recruits the histone deacetylases (HDACs) containing the nucleosome remodeling and deacetylase (NuRD) complex, to promote lung adenocarcinoma invasion and metastasis compared to ZEB1 acetyl-deficient. We further established that the stability of ZEB1 is integral to promoting these mesenchymal features. Our results suggest that ZEB1_K811ac regulates its dimerization and stability to promote lung adenocarcinoma via EMT. Future studies are focused on understanding the role of ZEB1 protein stability to recruit NuRD co-repressor and subsequently mediate tumorigenesis and metastasis.

Citation Format: Mabel G. Perez-Oquendo, Roxsan Manshouri, Don Gibbons. ZEB1 acetylation regulates dimerization and protein stability to promote lung adenocarcinoma progression and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 676.