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

• 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
• 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

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.

项目总结/摘要 非小细胞肺癌（NSCLC）是全球癌症相关死亡的主要原因，这是由于其具有治疗癌症的能力。 癌细胞转移上皮-间质转化（EMT）是转移的机制， 导致顶端-基底极性的丧失和特化上皮细胞接触以获得间质迁移 能力和侵略性。锌指E盒结合同源异型盒1（ZEB 1）转录抑制因子识别 并结合基因启动子区的E盒以抑制上皮基因如E-钙粘蛋白的表达。 ZEB 1招募转录辅抑制因子，事实上，我们最近报道ZEB 1与 组蛋白脱乙酰酶（HDAC）1和2含有核小体重塑和脱乙酰酶（NuRD）复合物， 调节其靶基因的转录。ZEB 1的预测分子量为125 kDa;然而， 几个研究组报道了观察到的分子量（约190-220 kDa）的不一致， 这已被归因于翻译后修饰（PTM）。到目前为止，分子调控 关联和功能，以及预测和观察到的ZEB 1分子之间的差异， 体重，目前还不清楚。我实验室以前的数据显示，小鼠和人NSCLC细胞系治疗后， I类HDAC抑制剂将ZEB 1的分子量从250 kDa降低到125 kDa。此外，通过 通过免疫共沉淀，我们证明了ZEB 1形成了依赖于I类HDAC的同源二聚体 活动因此，我们进行了质谱分析，并确定了一个新的PTM - K811乙酰化-这可能 调节ZEB 1二聚化、蛋白质相互作用和/或稳定性。因此，我们试图确定 通过产生ZEB 1乙酰基模拟物（K811 Q）和缺陷型（K811 R），二聚化中的ZEB 1乙酰化和活性 NSCLC细胞系中的突变体。我们假设ZEB 1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 ZEB 1二聚化和蛋白质稳定性受乙酰化调节，ii）评估是否同源二聚化 有助于ZEB 1/NuRD介导的转录抑制，iii）评估ZEB 1的功能作用 二聚化在NSCLC转移中的作用。我们的数据表明，乙酰基缺陷突变体（125 kDa）表现出 与野生型和乙酰化的ZEB 1（250 kDa）相比，减少的半衰期，这表明， 乙酰化阻碍蛋白质二聚化和稳定性。有趣的是，我们以前报道过ZEB 1优先 在NSCLC细胞系中与NuRD形成复合物;然而， ZEB 1二聚体与NuRD复合物调节其转移功能尚未确定。因此，委员会认为， 在唐·吉本斯博士和米歇尔·巴顿博士的指导下，我的目标是描述ZEB 1的贡献 二聚体/NuRD介导的转录抑制，并促进替代治疗药物的开发 靶向PTM的策略，最终目标是提高肺癌患者的生存率。