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

ZEB1二聚化在肺腺癌进展和转移中的调控及作用

基本信息

批准号：
10384885
负责人：
Mabel Perez-Oquendo
金额：
$ 3.47万
依托单位：
UNIVERSITY OF TX MD ANDERSON CAN CTR
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-15 至 2027-04-14
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10384885
关键词：
3-Dimensional Acetylation Address Apical Automobile Driving Back Binding Biological Biological Assay Cancer Etiology Cancer Patient Cancer cell line Cells Cessation of life Chemoresistance Chromatin Chromatin Remodeling Factor Co-Immunoprecipitations Complex Cycloheximide Data Deacetylase Detection Development Dimerization E-Cadherin Enzymes Epithelial Epithelial Cells Exhibits Genes Genetic Transcription Goals HDAC1 gene Half-Life Histone Deacetylase Histone Deacetylase Inhibitor Homeobox Homodimerization Human Hylobates Genus Immunoprecipitation Implant In Vitro Knowledge Ligation Lung Adenocarcinoma Lung nodule MG132 Malignant neoplasm of lung Mass Spectrum Analysis Mediating Mentorship Mesenchymal Molecular Molecular Weight Mus Neoplasm Metastasis Non-Small-Cell Lung Carcinoma Nucleosomes Pathologic Post-Translational Protein Processing Promoter Regions Proteasome Inhibitor Protein Biosynthesis Proteins RNA Regulation Reporting Research Research Proposals Resistance Role SEMA3F gene Site Specialized Epithelial Cell Therapeutic Transcription Repressor Translations Trichostatin A Zinc Fingers cancer cell dimer epithelial to mesenchymal transition gene repression genetic corepressor improved in vivo member metastatic process migration mimetics mutant novel overexpression promoter recruit subcutaneous tandem mass spectrometry therapeutic target transcription factor tumor tumor growth

项目摘要

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.

项目摘要/摘要非小细胞肺癌（NSCLC）是全球与癌症相关死亡的主要原因，因为癌细胞转移。上皮到间质转变（EMT）是转移的机制，导致顶端极性丧失和专门的上皮细胞接触以获取间质迁移能力和侵入性。锌指EBOXBINDING HONEOBOX 1（ZEB1）转录抑制剂识别并结合基因启动子区域的电子盒，以抑制上皮基因（例如E-钙粘蛋白）的表达。 Zeb1招募转录核心器，实际上，我们最近报道了Zeb1与组蛋白脱乙酰基酶（HDACS）1和2含有核小体重塑和脱乙酰基酶（NURD）复合物至调节其靶基因的转录。 Zeb1的预测分子量为125 kDa；然而，几个组报告了观察到的分子量（约190-220 kDa）的不一致，这归因于翻译后修改（PTM）。迄今为止，分子的调节关联和功能，以及预测和观察到的Zeb1分子之间的差异重量，仍然不清楚。我实验室的先前数据表明，鼠和人类NSCLC细胞系都处理过使用I类HDAC抑制剂，Zeb1的分子量从250 kDa降低至125 kDa。另外，通过共免疫沉淀，我们证明Zeb1形成了依赖于I类HDAC的同型二聚体活动。因此，我们进行了质谱法，并确定了一种新型的PTM -K811乙酰化 - 可能调节Zeb1二聚化，蛋白质相互作用和/或稳定性。因此，我们试图定义 Zeb1乙酰化在二聚化和活性中通过产生Zeb1乙酰基模拟物（K811Q）和缺乏（K811R） NSCLC细胞系中的突变体。我们假设Zeb1乙酰化通过NURD调节其二聚化复合物，随后促进NSCLC转移。我们将通过以下方式解决这一假设：i）确定是否是否 Zeb1二聚化和蛋白质稳定性受乙酰化的调节，ii）评估是否均二聚化有助于ZEB1/NURD介导的转录抑制，iii）评估Zeb1的功能效应 NSCLC转移中的二聚化。我们的数据表明，乙酰缺乏的突变体（125 kDa）表现出A 与野生型和乙酰化Zeb1（250 kDa）相比，半衰期减少了，这表明破坏了乙酰化阻碍蛋白质二聚化和稳定性。有趣的是，我们先前报道了Zeb1优先在NSCLC细胞系中与NURD形成复合物；但是，物理关联之间的重要性尚未确定具有NURD复合物调节其转移功能的Zeb1二聚体。因此，在Don Gibbons博士和Michelle Barton博士的指导下，我的目标是表征Zeb1的贡献二聚体/NURD介导的转录抑制，并促进替代治疗的发展针对PTM的策略的最终目标是改善肺癌患者的存活率。