Mechanisms of gene expression regulation in pancreatic cancer

胰腺癌基因表达调控机制

基本信息

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
项目类别：
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

项目摘要

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.

项目概要胰腺导管腺癌（PDAC）预计将成为全球第二大致命癌症未来10年。迫切需要研究 PDAC 转化的新机制，因为目前还没有目前存在有效的 PDAC 治疗方法。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起着至关重要的相互关联的作用。一个未阐明的关键方面转化背景下的基因调控是介导基因变化的分子机制。染色质重组。致癌 KRAS 诱导模型中的 ChIP-seq 和 ChIP-PCR 研究是关键 PDAC 中的起始因子，证明异染色质重新定位到核外围。这些叶片相关结构域 (LAD) 在 H3K9me2 中富集并位于核纤层。 ChIP 测序和 RNA-seq 发现 LAD 中活性增强子区域的丢失和 LAD 的下调分别是相关基因。研究调节LAD下游组装的机制致癌 KRAS 我们进行了多项分子和生化研究。我们进行了 BioID 筛选利用核纤层蛋白 A（已知与 LAD 相互作用的核纤层核心成分）作为诱饵。核纤层蛋白A BioID 鉴定出一种新型肌球蛋白，肌球蛋白 18a (MYO18a)，在致癌 KRAS 作用下富集于核层发信号。免疫细胞化学显示 MYO18a 的核定位增加，并且在 KRAS 激活后的层板。生化分析验证了 MYO18a-Lamin A 相互作用并证实 MYO18a 与染色质相互作用。 MYO18a 的 siRNA 敲低挽救了相关基因的表达具有致癌 KRAS 介导的 LAD。这些数据引导我们得出中心假设：MYO18a 充当 KRAS 下游效应子调节核纤层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 体外模型中检测 MYO18a 相关的 LAD。 LAD 装配将在 MYO18a 的存在/不存在。我们还将开发MYO18a基因工程小鼠模型来探索 MYO18a 和核 MYO18a 在 PDAC 发育和进展中的作用。对此进一步研究机制可以为转化过程中的基因调控提供有价值的见解，并将识别潜在的药物靶标。