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

项目摘要/摘要 非小细胞肺癌(NSCLC)是全球癌症相关死亡的主要原因，其原因是 癌细胞转移。上皮向间充质转化(EMT)是肿瘤转移的一种机制。 导致根尖-基底极性的丧失和特殊的上皮细胞接触以获得间充质迁移 能力和侵入性。锌指E盒结合同源框1(ZEB1)转录抑制因子识别 并结合基因启动子区域的E盒，抑制E-钙粘附素等上皮基因的表达。 ZEB1招募转录辅阻遏子，事实上，我们最近报道了ZEB1与 组蛋白脱乙酰酶(HDACs)1和2含有核小体重塑和脱乙酰酶(NuRD)复合体 调节其目标基因的转录。ZEB1的预测分子量为125 kDa；然而， 几个小组报告了观察到的分子量不一致(约190-220 kDa)， 这被归因于翻译后修改(PTM)。到目前为止，分子生物学的调控 关联和功能，以及预测和观察到的ZEB1分子之间的差异 重量，目前仍不清楚。之前来自我的实验室的数据显示，小鼠和人类的NSCLC细胞株 使用I类HDAC抑制剂，ZEB1的相对分子质量从250 kDa降低到125 kDa。此外，通过 免疫共沉淀，我们证明了ZEB1形成一种依赖于I类HDAC的同源二聚体 活动。因此，我们进行了质谱分析，并确定了一种新的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乙酰化反应中的二聚化及其生成模拟(K811Q)和缺失(K811R)的活性 非小细胞肺癌细胞系中的突变型。我们假设ZEB1乙酰化通过NuRD调节其二聚化。 复合体，从而促进非小细胞肺癌转移。我们将通过以下方式解决这一假设：i)确定 ZEB1二聚化和蛋白质稳定性受乙酰化调控，II)评估同源二聚化是否 参与ZEB1/NuRD介导的转录抑制，III)评估ZEB1的功能效应 二聚体在非小细胞肺癌转移中的作用我们的数据表明，缺乏乙酰基的突变体(125 KDa)表现出 与野生型和乙酰化ZEB1(250 KDa)相比，半衰期减少，表明 乙酰化阻碍了蛋白质的二聚化和稳定性。有趣的是，我们之前报道过ZEB1优先 在非小细胞肺癌细胞系中与NuRD形成复合体；然而，物理关联的意义在于 ZEB1二聚体与NuRD复合体一起调节其转移功能尚未确定。因此， 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ZEB1的贡献 二聚体/NuRD介导的转录抑制和促进替代治疗的发展 以提高肺癌患者存活率为最终目标的PTMS策略。

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.