Defining Mechanisms of Transformation Driven By the Zinc Finger Transcription Factor PLAGL2 in the Intestinal Epithelium

定义肠上皮中锌指转录因子 PLAGL2 驱动的转化机制

基本信息

批准号：
10591499
负责人：
DEBORAH C. RUBIN
金额：
$ 35.31万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-01 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10591499
关键词：
APC gene APC mutation ASCL2 gene Adenocarcinoma Adult Alleles Automobile Driving Binding Sites Biological Assay Cancerous Cell Culture Techniques Cell Differentiation process Cells Clustered Regularly Interspaced Short Palindromic Repeats Colon Colonic Neoplasms Colonic Polyps Colorectal Cancer Colorectal Neoplasms Data Distal Embryonic Development Epithelial Cells Epithelium Familial Adenomatous Polyposis Syndrome Family Genes Genetic Transcription Genetically Engineered Mouse Genomics Growth Human In Vitro Intestines Investigation Knock-out LoxP-flanked allele MADH4 gene Maintenance Malignant - descriptor Malignant Neoplasms Manuscripts Mediator Messenger RNA MicroRNAs Modeling Mucous Membrane Mus Mutagenesis Mutate Mutation Non-Malignant Oncogenes Oncogenic Organoids PTEN gene Pathway interactions Patients Play Pongidae Premalignant Change Process Proliferating Publishing RNA Regulatory Element Reporter Reporting Repression Role Signal Pathway TP53 gene The Cancer Genome Atlas Tissues Transforming Growth Factor beta Tumor Burden Tumor stage Up-Regulation WNT Signaling Pathway Work Zinc Fingers adenoma cell transformation colon cancer cell line colon tumorigenesis colonic crypt gain of function gene repression in vivo intestinal epithelium intestinal tumorigenesis loss of function malignant phenotype mouse model novel novel therapeutics overexpression premalignant prevent programs self-renewal stem stem cell biomarkers stem cell fate stem cells targeted treatment transcription factor tumor tumorigenesis vector

项目摘要

PROJECT SUMMARY/ABSTRACT This project will focus on understanding mechanisms of intestinal epithelial cell (IEC) transformation. We define early features of transformation as the augmentation of proliferation, stem cell fate, and repression of IEC differentiation. Here, we seek to understand the role of a specific transcription factor (PLAGL2) we have recently identified as a target repressed by Let-7 microRNAs as a part of a recently published manuscript in Stem Cell Reports, showing that PLAGL2 drives stem cell fate. Our previous studies indicate that Let-7 is a potent repressor of tumorigenesis and intestinal stem cell fate. Many Let-7 targets have been implicated as oncogenes in multiple tissues, including MYC and RAS, but in our models of Let-7 manipulation in the intestine, we do not find significant effects on these targets. We present evidence that our newly identified Let- 7 target, PLAGL2, is commonly up-regulated (>60%) in pre-cancerous and cancerous colorectal tumors in humans, with expression inversely proportional to Let-7 miRNAs. This potentially represents a major pathway of tumorigenesis as both Let-7 and target mRNAs are deregulated in the majority of colorectal cancers (CRC), and is thus highly relevant. In human CRC cell lines we find that PLAGL2 is necessary for driving a malignant phenotype, while in normal IEC organoids PLAGL2 can drive early features of transformation. First, to identify roles for PLAGL2 in IEC transformation we will look at the potential for this factor to drive tumorigenesis through targeted over-expression in the intestine, in the context of Let-7 depletion through LIN28B over- expression. Using a newly generated floxed allele, we will also determine whether PLAGL2 is required for driving early stages of IEC transformation in this model. Second, we will use our novel CRISPR somatic mutagenesis model of intestinal tumorigenesis to examine interaction and cooperation of PLAGL2 with canonical CRC oncogenic pathways. This model randomly mutates the tumor suppressors Apc, Pten, Smad4, and Trp53 to generate adenomas and adenocarcinomas, which allows us to study PLAGL2 across an array of malignant stages, tumor types, in the context of different, but defined, mutations. Third, we will explore the roles of relevant PLAGL2 transcriptional targets, including target genes we have already identified, in driving early processes of transformation. Through new assays and reporters in organoids, we will also characterize Wnt signaling activity in the clonogenic cell of origin that is mobilized by aberrant PLAGL2 expression. These investigations will likely illuminate how this newly discovered Let-7 target, PLAGL2, drives pre-malignant changes and tumorigenesis. Considering its widespread up-regulation in pre-cancerous and cancerous growths in the intestine, PLAGL2 may represent a keystone target for therapeutic inhibition. Additionally, given the potent effects on stem cell fate that we see driven by PLAGL2, this factor may also play a key role in the early definition, or later maintenance, of intestinal stem cells.

项目概要/摘要该项目将重点了解肠上皮细胞（IEC）转化的机制。我们将转化的早期特征定义为增殖、干细胞命运的增强和 IEC 差异化。在这里，我们试图了解我们拥有的特定转录因子 (PLAGL2) 的作用最近被确定为被 Let-7 microRNA 抑制的目标，作为最近发表的手稿的一部分干细胞报告显示 PLAGL2 驱动干细胞命运。我们之前的研究表明 Let-7 是肿瘤发生和肠道干细胞命运的有效抑制因子。许多 Let-7 目标被认为是多种组织中的癌基因，包括 MYC 和 RAS，但在我们的 Let-7 操作模型中肠道，我们没有发现对这些目标有显着影响。我们提供的证据表明我们新确定的 Let- 7 靶标 PLAGL2 在癌前和癌性结直肠肿瘤中通常上调 (>60%) 人类，表达量与 Let-7 miRNA 成反比。这可能代表了一条主要途径由于 Let-7 和靶标 mRNA 在大多数结直肠癌 (CRC) 中均失调，因此会影响肿瘤发生，因此具有高度的相关性。在人类结直肠癌细胞系中，我们发现 PLAGL2 对于驱动恶性结直肠癌是必需的。表型，而在正常的 IEC 类器官中，PLAGL2 可以驱动早期特征的转化。首先，要识别 PLAGL2 在 IEC 转化中的作用我们将研究该因素驱动肿瘤发生的潜力在通过 LIN28B 过度表达 Let-7 的情况下，通过肠道内的靶向过度表达表达。使用新生成的 floxed 等位基因，我们还将确定 PLAGL2 是否是必需的推动该模型中 IEC 转型的早期阶段。其次，我们将使用我们的新型 CRISPR 体细胞肠道肿瘤发生的诱变模型，用于检查 PLAGL2 与典型的 CRC 致癌途径。该模型随机突变肿瘤抑制因子 Apc、Pten、Smad4、和 Trp53 产生腺瘤和腺癌，这使我们能够跨一系列研究 PLAGL2 恶性阶段、肿瘤类型，在不同但明确的突变背景下。第三，我们将探讨相关 PLAGL2 转录靶标（包括我们已经确定的靶基因）在驱动中的作用转型的早期过程。通过类器官中的新测定和报告基因，我们还将表征克隆源细胞中的 Wnt 信号传导活性由异常的 PLAGL2 表达动员。这些调查可能会阐明这个新发现的 Let-7 靶标 PLAGL2 如何驱动癌前病变变化和肿瘤发生。考虑到其在癌前期和癌中的广泛上调 PLAGL2 可能是抑制肠道内生长的关键靶点。另外，给定我们看到 PLAGL2 驱动对干细胞命运的有效影响，该因子也可能在肠道干细胞的早期定义或后期维护。