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.

项目总结/摘要 本项目将侧重于了解肠上皮细胞（IEC）转化的机制。我们 将转化的早期特征定义为增殖的增强，干细胞的命运，以及对 IEC区分。在这里，我们试图了解一个特定的转录因子（PLAGL 2）的作用，我们有 最近被确定为Let-7 microRNA抑制的靶点，作为最近发表的手稿的一部分， 干细胞报告，显示PLAGL 2驱动干细胞命运。我们以前的研究表明Let-7是一种 肿瘤发生和肠干细胞命运的有效阻遏物。许多Let-7目标被牵连， 在多个组织中的癌基因，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显着影响。我们提出的证据表明，我们新发现的Let- 7靶点PLAGL 2在大肠癌患者的癌前和癌性结肠直肠肿瘤中通常上调（>60%）。 人，其表达与Let-7 miRNA成反比。这可能代表了一个主要途径， 由于Let-7和靶mRNA在大多数结直肠癌（CRC）中失调， 因此是高度相关的。在人CRC细胞系中，我们发现PLAGL 2是驱动恶性肿瘤细胞增殖所必需的。 在正常的IEC类器官中，PLAGL 2可以驱动转化的早期特征。首先确定 PLAGL 2在IEC转化中的作用，我们将研究该因子驱动肿瘤发生的潜力 通过在肠中靶向过表达，在通过LIN 28 B过表达的Let-7消耗的背景下， 表情使用新产生的floxed等位基因，我们还将确定PLAGL 2是否是 推动IEC转型的早期阶段。第二，我们将使用我们的新型CRISPR体细胞， 肠肿瘤发生的诱变模型，以检查PLAGL 2与 典型的CRC致癌通路。该模型随机突变肿瘤抑制因子Apc、Pten、Smad 4， 和Trp 53产生腺瘤和腺癌，这使我们能够在一系列的细胞中研究PLAGL 2。 恶性阶段，肿瘤类型，在不同的背景下，但定义，突变。第三，我们将探讨 相关PLAGL 2转录靶点的作用，包括我们已经确定的靶基因，在驱动 早期的转型过程。通过类器官中的新检测和报告，我们还将表征 由异常PLAGL 2表达动员的起源克隆细胞中的Wnt信号传导活性。这些 研究可能会阐明这种新发现的Let-7靶标PLAGL 2如何驱动癌前病变， 变化和肿瘤发生。考虑到它在癌前和癌组织中的广泛上调， 对于肠中的生长，PLAGL 2可能代表治疗抑制的关键靶标。此外，鉴于 我们看到PLAGL 2对干细胞命运的强大影响，这个因子也可能在细胞凋亡中发挥关键作用。 肠干细胞的早期定义或后期维护。