Mechanisms of gene expression regulation in pancreatic cancer

胰腺癌基因表达调控机制

• 批准号: 10743387
• 负责人: David R Pease
• 金额: $ 4.92万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-09 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10743387
• 关键词: Automobile Driving; Binding; Biochemical; Biological; Cancer Biology; Cell membrane; ChIP-seq; Chromatin; Confocal Microscopy; DNA Sequence Alteration; Data; Development; Disease; Down-Regulation; Enhancers; Epigenetic Process; Event; Foundations; Gene Expression; Gene Expression Profiling; Gene Expression Regulation; Genes; Genetic Engineering; Genome; Goals; Golgi Apparatus; Growth; Growth and Development function; Heterochromatin; Investigation; KRAS oncogenesis; KRAS2 gene; Lamin Type A; Lead; Lesion; Lysine; Malignant Neoplasms; Malignant neoplasm of pancreas; Maps; Mediating; Modeling; Molecular; Morphology; Mus; Mutate; Mutation; Myosin ATPase; Nuclear; Nuclear Lamina; Oncogenes; Oncogenic; Outcome; Pancreatic Ductal Adenocarcinoma; Pathway interactions; Patients; Peptide Initiation Factors; Phase; Play; Positioning Attribute; Postdoctoral Fellow; Process; Proteins; Regulation; Research; Role; Signal Pathway; Signal Transduction; Small Interfering RNA; Survival Rate; Testing; Time; Training; Transcriptional Regulation; Walking; cellular imaging; conditional mutant; druggable target; effective therapy; epigenetic silencing; epigenomics; experimental study; immunocytochemistry; in vitro Model; in vivo; insight; knock-down; live cell imaging; loss of function; mouse model; mutant; neoplastic; novel; pancreatic ductal adenocarcinoma cell; pancreatic ductal adenocarcinoma model; programs; protein protein interaction; screening; single-cell RNA sequencing; therapeutic target; transcriptome sequencing; transcriptomics; tumor

PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDAC) is predicted to become the second deadliest cancer within the next 10 years. Investigations into novel mechanisms of PDAC transformation are urgently needed as no effective PDAC treatment currently exists. Transcriptional regulation during transformation is multifaceted with genetic mutations and epigenetic alterations playing vital interconnected roles. A key unelucidated facet of gene regulation in the context of transformation is the molecular mechanism which mediates changes in chromatin reorganization. ChIP-seq and ChIP-PCR studies in an inducible model of oncogenic KRAS, a key initiating factor in PDAC, demonstrate a relocation of heterochromatin to the nuclear periphery. These lamina associated domains (LADs) are enriched in H3K9me2 and positioned at the nuclear lamina. ChIP-seq and RNA-seq identified loss of active enhancer regions incorporated into LADs and downregulation of LAD associated genes, respectively. To investigate the mechanism regulating the assembly of LADs downstream of oncogenic KRAS we performed several molecular and biochemical studies. We conducted a screening BioID utilizing Lamin A, a core component of the nuclear lamina known to interact with LADs, as bait. Lamin A BioID identified a novel myosin, Myosin 18a (MYO18a), enriched at the nuclear lamina under oncogenic KRAS signaling. Immunocytochemistry revealed increased nuclear localization of MYO18a and enrichment at the lamina upon KRAS activation. Biochemical analysis validated MYO18a-Lamin A interaction and confirmed MYO18a interaction with chromatin. siRNA knockdown for MYO18a rescued expression of genes associated with oncogenic KRAS-mediated LADs. These data lead us to our central hypothesis that MYO18a acts as a downstream effector of KRAS to modulate chromatin positioning at the nuclear lamina to silence oncogenic gene expression. To test this hypothesis, we will conduct ChIP-seq and RNA-seq experiments in parallel to detect MYO18a associated LADs in in vitro models of PDAC. LAD assembly will then be investigated in the presence/absence of MYO18a. We will also develop MYO18a genetic engineered mouse models to explore the role of MYO18a and nuclear MYO18a in PDAC development and progression. Further study into this mechanism can provide valuable insight into gene regulation in the transformation process and will identify potentially druggable targets.

项目摘要 胰腺导管腺癌（PDAC）预计将成为美国第二大致命癌症。 未来10年目前，研究PDAC转化的新机制是迫切需要的， 目前存在有效的PDAC治疗。转化过程中的转录调控是多方面的， 基因突变和表观遗传改变发挥着至关重要的相互关联的作用。一个关键的未阐明的方面， 在转化过程中的基因调控是介导转化过程中的变化的分子机制。 染色质重组在致癌KRAS的诱导模型中的ChIP-seq和ChIP-PCR研究， 作为PDAC中的起始因子，证明异染色质向核周边的重新定位。这些叶片 相关结构域（LAD）富含H3 K9 me 2并位于核纤层。ChIP-seq和 RNA-seq鉴定了掺入LAD的活性增强子区域的丢失和LAD的下调 相关基因。目的：探讨调节前降支下游聚集的机制。 我们进行了几项分子和生物化学研究。我们进行了生物识别 利用核纤层蛋白A作为诱饵，核纤层蛋白A是已知与LAD相互作用的核纤层的核心组分。核纤层蛋白A生物识别 发现了一种新的肌球蛋白，肌球蛋白18 a（MYO 18 a），在致癌KRAS下富集于核纤层 信号免疫细胞化学显示MYO 18 a的核定位增加， KRAS激活后的纤层。生化分析验证了MYO 18 a-Lamin A相互作用，并证实了MYO 18 a-Lamin A相互作用。 MYO 18 a与染色质的相互作用。MYO 18 a的siRNA敲低挽救了相关基因的表达 致癌KRAS介导的LAD这些数据使我们得出了我们的中心假设，即MYO 18 a作为一种 KRAS的下游效应子，以调节核纤层处的染色质定位以沉默致癌基因 基因表达。为了验证这一假设，我们将同时进行ChIP-seq和RNA-seq实验， 在PDAC的体外模型中检测MYO 18 a相关的LAD。然后，将在 MYO 18 a的存在/不存在。我们还将开发MYO 18 a基因工程小鼠模型，以探索 MYO 18 a和核MYO 18 a在PDAC发生和进展中的作用。对此进一步研究 机制可以提供有价值的洞察基因调控在转化过程中，并将确定 潜在的药物目标