Regulation and Function of ZEB1 Dimerization in Lung Adenocarcinoma Progression and Metastasis

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Project Summary/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. Epithelial-to-mesenchymal transition (EMT) is a mechanism for metastasis, which results in a loss of apical-basal polarity and specialized epithelial cell contacts to acquire mesenchymal migratory capacity and invasiveness. The Zinc finger E-box binding homeobox 1 (ZEB1) transcription repressor recognizes and binds E-boxes of gene promoter regions to suppress the expression of epithelial genes such as E-cadherin. ZEB1 recruits transcriptional corepressors and, in fact, we have recently reported that ZEB1 interacts with histone deacetylases (HDACs) 1 and 2 containing nucleosome remodeling and deacetylase (NuRD) complex to regulate the transcription of their target genes. ZEB1 has a predicted molecular weight of 125 kDa; however, several groups have reported inconsistencies in the observed molecular weight (approximately 190-220 kDa), which has been attributed to post-translational modifications (PTMs). To date, the regulation of molecular associations and functions, as well as the discrepancy between the predicted and observed ZEB1 molecular weight, is still unclear. Previous data from my lab shows that both murine and human NSCLC cell lines treated with class I HDAC inhibitors reduce the molecular weight of ZEB1 from 250 kDa to 125 kDa. Additionally, through co-immunoprecipitation, we demonstrated that ZEB1 forms a homodimer that is dependent on class I HDAC activity. Therefore, we performed mass spectrometry and identified a novel PTM - K811 acetylation - that may regulate ZEB1 dimerization, protein interactions, and/or stability. Consequently, we sought to define the role of ZEB1 acetylation in dimerization and activity by generating ZEB1 acetyl mimetic (K811Q) and deficient (K811R) mutants in NSCLC cell lines. We hypothesize that ZEB1 acetylation regulates its dimerization via the NuRD complex, which subsequently promotes NSCLC metastasis. We will address this hypothesis by: i) determining if ZEB1 dimerization and protein stability are regulated by acetylation, ii) evaluating whether homodimerization contributes to ZEB1/NuRD-mediated transcriptional repression, iii) assessing the functional effect of ZEB1 dimerization in NSCLC metastasis. Our data demonstrated that the acetyl-deficient mutant (125 kDa) exhibits a decreased half-life compared to wild-type and acetylated ZEB1 (250 kDa), suggesting that disruption of acetylation hinders protein dimerization and stability. Intriguingly, we previously reported that ZEB1 preferentially forms a complex with NuRD in NSCLC cell lines; however, the significance of the physical association between ZEB1 dimers with the NuRD complex to regulate its metastatic function has not been identified. Accordingly, under the mentorship of Dr. Don Gibbons and Dr. Michelle Barton, I aim to characterize the contribution of ZEB1 dimer/NuRD-mediated transcriptional repression and to facilitate the development of alternative therapeutic strategies targeting PTMs with the ultimate goal of improving the survival of lung cancer patients.