Abstract LBA023: Regulation and function of ZEB1 dimerization in lung adenocarcinoma progression and metastasis

Abstract

Abstract 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 the 393P lung adenocarcinoma cell line. 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 28.03hrs to 44.37hrs and acetyl-mimetic from 19.43hrs to 41.25hrs, as well as 125kDa ZEB1 acetyl-deficient from 9.38hrs to 12.70hrs. 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 by Chromatin Immunoprecipitation. 250kDa ZEB1 WT and acetyl-mimetic promotes 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 PTMs (acetylation and ubiquitination) regulate ZEB1 to promote its stability and progression of lung adenocarcinoma via EMT. Future studies are focused on further understanding the role of ZEB1 dimerization to recruit co-repressors and subsequently mediate tumorigenesis, invasion and metastasis Citation Format: Mabel Perez-Oquendo, Roxsan Manshouri, Don L. Gibbons. Regulation and function of ZEB1 dimerization in lung adenocarcinoma progression and metastasis [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA023.