Interplay of IMP1 and autophagy in intestinal barrier function and tumorigenesis

 描述（由申请人提供）：肠上皮细胞在增殖和分化之间具有微妙的平衡。两个干细胞群驻留在隐窝中，并参与稳态和对应激源的适应性反应，如炎症（如炎症性肠病）和肿瘤发生（结直肠癌，CRC）。很明显，上皮细胞稳态异常和肿瘤发生之间存在相互作用。最近发现的促进或抑制CRC的基因突变指导了治疗策略的发展，但CRC死亡率仍然升高，这表明涉及其他途径或分子介质。该提案将研究一种可能有助于CRC启动或进展的新途径，并为肠上皮稳态和屏障功能提供新的见解。Igf 2-mRNA结合蛋白1（IMP 1）在胚胎发育和癌症期间高度表达。IMP 1稳定mRNA，包括Igf 2、cMyc和Kras。我们的研究小组最近证明，在CRC异种移植模型中，IMP 1的过表达增强了肿瘤生长，癌症干细胞标志物表达，并降低了E-钙粘蛋白（对细胞-细胞接触很重要）。我们也有新的初步数据表明，IMP 1在CRC细胞中的调节改变了自噬蛋白的表达。自噬是细胞在应激情况下（如细胞损伤、炎症或癌症）分解细胞器或细胞内成分的过程。自噬可以是肿瘤抑制的或致癌的。IMP 1在调节自噬在体内平衡和癌症中的作用是未知的。还不清楚表达IMP 1的细胞是否直接启动肿瘤形成，或通过直接增强自噬、EMT或细胞分化相关途径促进进展。我们推测IMP 1通过调节自噬介导肠屏障功能和肿瘤发生。我们将通过两个相互关联的具体目标来检验这一假设。在目标1中，我们将使用细胞系和遗传小鼠模型来定义IMP 1和自噬在正常屏障功能和癌前炎症中的功能作用。具体而言，我们将利用慢病毒系统产生具有IMP 1过表达或敲低的Caco-2细胞系，从WT; Imp 1fl/fl或VillinCre; Imp 1fl/fl小鼠产生肠类，并使用葡聚糖硫酸钠（DSS）在WT; Imp 1fl/fl或VC; Imp 1fl/fl小鼠中诱导急性肠损伤。将使用Cyto-ID、培养细胞中的跨上皮抗性测量和体内渗透性测定来询问所有系统对自噬的影响。在目标2中，我们将定义 IMP 1和自噬在肿瘤发生和发展中的作用这将在WT; Imp 1fl/fl或VC; Imp 1fl/fl小鼠中使用炎症相关癌症的AOM/DSS模型来实现，并且将通过将我们的IMP 1报告基因模型（Imp 1 EGFP;DTR;CreER）与Apcflfl小鼠交叉以检查肿瘤起始来评估IMP 1阳性细胞在肿瘤起始中的作用。总之，这些研究将为IMP 1在屏障功能和肿瘤发生/进展中的作用提供新的见解。这些结果将为潜在的翻译应用提供一个平台。

 DESCRIPTION (provided by applicant): The intestinal epithelium has an exquisite equilibrium between proliferation and differentiation. Two stem cell populations reside in the crypts and are involved in homeostasis and adaptive responses to stressors, such as inflammation (as in inflammatory bowel disease) and tumorigenesis (colorectal cancer, CRC). It is clear there is interplay between aberrations in epithelial homeostasis and tumorigenesis. Recent discoveries of genetic mutations that promote or suppress CRC have guided development of therapeutic strategies, yet CRC deaths remain elevated, suggesting that additional pathways or molecular mediators are involved. This proposal will examine a new pathway that may contribute to CRC initiation or progression, as well as provide new insights into intestinal epithelial homeostasis and barrier function. Igf2-mRNA binding protein 1 (IMP1) is expressed highly during embryonic development and cancer. IMP1 stabilizes mRNAs, including Igf2, cMyc, and Kras. Our group recently demonstrated that overexpression of IMP1 enhances tumor growth, cancer stem cell marker expression, and decreased E-cadherin (important for cell-cell contacts), in CRC xenograft models. We also have novel preliminary data suggesting that modulation of IMP1 in CRC cells alters expression of autophagic proteins. Autophagy is a process by which cells breakdown organelles or intracellular components during situations of stress, such as cellular damage, inflammation, or cancer. Autophagy can be tumor suppressive or oncogenic. The role of IMP1 in modulating autophagy in homeostasis and cancer is unknown. It is also unknown whether IMP1-expressing cells directly initiate tumor formation, or promote progression through direct augmentation of autophagy-, EMT- or cell differentiation-associated pathways. We hypothesize that IMP1 mediates intestinal barrier function and tumorigenesis via modulation of autophagy. We will test this hypothesis through two interrelated Specific Aims. In Aim 1 we will use cell lines and genetic mouse models to define the functional roles of IMP1 and autophagy in normal barrier function and precancerous inflammation. Specifically, we will utilize lentiviral systems to generate Caco-2 cell lines with IMP1 overexpression or knockdown, generate intestinal enteroids from WT; Imp1fl/fl or VillinCre; Imp1fl/fl mice, and induce acute intestinal damage in WT; Imp1fl/fl or VC; Imp1fl/fl mice using dextran sodium sulftate (DSS). All systems will be interrogated for effects on autophagy using Cyto-ID, trans-epithelial resistance measures in cultured cells, and in vivo permeability assays. In Aim 2 we will define the functional roles of IMP1 and autophagy in tumor initiation and progression. This will be achieved using the AOM/DSS model of inflammation-associated cancer in WT; Imp1fl/fl or VC;Imp1fl/fl mice and the role of IMP1-positive cells in tumor initiation will be assessed by crossing our IMP1 reporter model (Imp1EGFP;DTR;CreER) with Apcflfl mice to examine tumor initiation. In aggregate, these studies will provide new insights into roles of IMP1 in barrier function and tumor initiation/progression. These results will provide a platform for potential translational applications.