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

项目摘要/摘要 本项目将致力于了解肠上皮细胞(IEC)转化的机制。我们 将转化的早期特征定义为促进增殖、干细胞命运和抑制 IEC分化。在这里，我们试图了解我们拥有的一种特定转录因子(PLAGL2)的作用 作为最近发表的一篇手稿的一部分，最近被确定为被let-7 microRNAs抑制的靶标 干细胞报告显示，PLAGL2决定了干细胞的命运。我们之前的研究表明，let-7是一种 肿瘤发生和肠道干细胞命运的有效抑制因子。许多let-7目标被牵连为 多种组织中的癌基因，包括MYC和RAS，但在我们的let-7操纵模型中 肠道，我们没有发现对这些靶点有显著影响。我们提供证据证明我们最新确认的- 7靶基因PLAGL2在癌前病变和结直肠癌中通常上调(60%)。 人类，其表达与let-7 miRNAs成反比。这可能代表着一条主要的途径 在大多数结直肠癌(CRC)中，由于let-7和靶向mRNAs都被解除调控， 因此具有很高的相关性。在人类结直肠癌细胞系中，我们发现PLAGL2是驱动恶性肿瘤所必需的 在正常的IEC中，PLAGL2可以驱动早期特征的转化。首先，要确定 PLAGL2在IEC转化中的作用我们将研究该因子驱动肿瘤发生的可能性 通过在肠道中靶向过度表达，在通过Lin28b过度耗尽let-7的背景下- 表情。使用新产生的等位基因，我们还将确定PLAGL2是否需要 在此模式中推动IEC转型的早期阶段。第二，我们将使用我们的新型CRISPR体细胞 检测PLAGL2与PLAGL2相互作用和协同作用的肠道致癌突变模型 典型的结直肠癌致癌途径。该模型随机突变肿瘤抑制基因APC、Pten、Smad4、 和Trp53来产生腺瘤和腺癌，这使我们能够研究PLAGL2在一系列 恶性阶段，肿瘤类型，在不同的背景下，但明确的，突变。第三，我们将探索 相关的PLAGL2转录靶基因，包括我们已经确定的靶基因，在驾驶中的作用 转型的早期过程。通过新的化验和有机化合物中的记者，我们还将表征 由PLAGL2异常表达所动员的起源克隆细胞中的WNT信号活性。这些 研究可能会阐明这个新发现的let-7靶点PLAGL2是如何推动癌前病变的 变化和肿瘤发生。考虑到它在癌前病变和癌组织中普遍上调 在肠道中的生长，PLAGL2可能是治疗抑制的关键靶点。此外，给出了 我们看到PLAGL2对干细胞命运的强大影响，这个因素也可能在 肠道干细胞的早期定义，或后来的维持。

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.