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

• 批准号: 10368956
• 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
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10368956
• 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; Colonic Neoplasms; Colonic Polyps; Colorectal Cancer; Colorectal Neoplasms; Data; Distal; Embryonic Development; Epithelial; Epithelial Cells; Familial Adenomatous Polyposis Syndrome; Family; Genes; Genetic Transcription; Genetically Engineered Mouse; Genomics; Growth; Human; In Vitro; Intestines; Investigation; Knock-out; Lead; LoxP-flanked allele; MADH4 gene; Maintenance; Malignant - descriptor; Malignant Neoplasms; Manuscripts; Mediator of activation protein; 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; Publishing; RNA; Regulation; 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; 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

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.

项目摘要/摘要 该项目将集中于理解肠上皮细胞（IEC）转化的机制。我们 将转化的早期特征定义为增强，干细胞命运的增强和抑制 IEC差异化。在这里，我们试图了解特定转录因子（plagl2）的作用 最近被视为被let-7 microRNA被抑制为目标的目标 干细胞报告，表明损伤驱动干细胞命运。我们以前的研究表明，Let-7是 肿瘤发生和肠道干细胞命运的有效抑制剂。许多Let-7目标被认为是 多种组织中的致癌基因，包括MYC和RAS，但在我们的Let-7操纵模型中 肠，我们没有发现对这些目标的重大影响。我们提供证据表明，我们新确定的租约 7靶标，plagl2通常在癌前和癌性结直肠肿瘤中被上调（> 60％） 人类，表达与Let-7 miRNA成反比。这可能代表了一条主要途径 在大多数结直肠癌（CRC）中，肿瘤发生为let-7和靶标mRNA。 因此非常相关。在人类CRC细胞系中，我们发现plagl2对于驾驶恶性剂是必需的 表型，虽然在正常的IEC器官中，plagl2可以推动转化的早期特征。首先，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 通过肠道中的靶向过表达，在通过LIN28B过度耗尽的情况下 表达。使用新生成的FloxErele，我们还将确定是否需要plagl2 在此模型中驱动IEC转换的早期阶段。第二，我们将使用我们的小说CRISPR躯体 肠道肿瘤发生的诱变模型，以检查plagl2的相互作用和合作 规范CRC致癌途径。该模型随机突变肿瘤抑制剂APC，PTEN，SMAD4， 和TRP53生成腺瘤和腺癌，这使我们能够在一系列阵列中研究plagl2 在不同但定义的突变的背景下，恶性阶段，肿瘤类型。第三，我们将探索 相关的PLAGL2转录靶标的角色，包括我们已经确定的靶基因，在驱动 转型的早期过程。通过细节的新测定和记者，我们还将表征 Wnt信号传导活性在原始细胞中被异常的plagl2表达动员。这些 调查可能会阐明这种新发现的let-7目标，plagl2，驱动预防剂 变化和肿瘤发生。考虑到其在癌前和癌症中的广泛上调 肠道中的生长可能是治疗抑制的基石靶标。另外，给予 我们看到的对干细胞命运的有效影响，我们看到的是损伤的驱动，该因素也可能在 早期定义或较晚的肠道干细胞维护。