Abstract 2477: 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. Epithelial-to-mesenchymal transition (EMT) is a mechanism for metastasis, which changes polarized epithelial cells into mesenchymal cells with enhanced motility and invasiveness. 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. Overexpression of each ZEB1 mutant fused with GFP demonstrated nuclear localization consistent with wild-type (WT) ZEB1. Interestingly, 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 and promotes migration and invasion in vitro compared to ZEB1 acetyl-deficient. We further established that the control GFP vector grew as non-invasive in 3D culture, but ZEB1 (WT and acetylation) colonies displayed an elongated invasive response associated with Rac1 and Fak signaling activation. Conversely, disruption of acetylation produced rounded invasive structures associated with RhoA expression. Rac1/RhoA small GTPases are key regulators of the mesenchymal or amoeboid mode of migration respectively as well as the transition between each mode (MAT and AMT). Rac1 inhibitor and RhoA inhibitor markedly reduced EMT characteristics in the ZEB1 acetyl-mimetic and acetyl-deficient mutants, respectively, suggesting that EMT may contribute to MAT. Combined, our results suggest that PTM regulates ZEB1 to promote its stability and progression of lung adenocarcinoma via EMT. Future studies are focused on further understanding the role of ZEB1 acetylation in vivo. However, the fact that acetylation contributes to ZEB1-mediated metastasis demonstrates its potential for use in the treatment of lung cancer. Citation Format: Mabel G. Perez-Oquendo, Michelle Barton, Don L. Gibbons. Regulation and function of ZEB1 dimerization in lung adenocarcinoma progression and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2477.