Interplay of IMP1 and autophagy in intestinal barrier function and tumorigenesis

 描述(申请人提供)：肠上皮在增殖和分化之间有一个微妙的平衡。隐窝中有两个干细胞群体，它们参与对应激因素的动态平衡和适应性反应，如炎症(如炎症性肠病)和肿瘤形成(结直肠癌)。很明显，上皮细胞动态平衡的异常与肿瘤的发生之间存在相互作用。最近发现的促进或抑制结直肠癌的基因突变指导了治疗策略的发展，但结直肠癌死亡率仍然居高不下，这表明有更多的途径或分子介质参与其中。这项建议将研究一种新的途径，可能有助于结直肠癌的启动或进展，以及提供对肠上皮稳态和屏障功能的新见解。胰岛素样生长因子2-信使核糖核酸结合蛋白1(IMP1)在胚胎发育和肿瘤过程中高表达。IMP1稳定包括Igf2、cMyc和Kras在内的mRNAs。我们的研究小组最近证明，在结直肠癌异种移植模型中，IMP1的过表达增强了肿瘤生长、肿瘤干细胞标记物的表达，并降低了E-钙粘附素(对细胞-细胞接触很重要)。我们也有新的初步数据表明，在结直肠癌细胞中IMP1的调节改变了自噬蛋白的表达。自噬是细胞在应激状态下分解细胞器或细胞内成分的过程，如细胞损伤、炎症或癌症。自噬可以是肿瘤抑制或致癌。IMP1在动态平衡和癌症中调节自噬的作用尚不清楚。目前还不清楚表达IMP1的细胞是直接启动肿瘤形成，还是通过直接增强自噬、EMT或细胞分化相关途径促进进展。我们假设IMP1通过调节自噬来调节肠道屏障功能和肿瘤的发生。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确定IMP1和自噬在正常屏障功能和癌前炎症中的功能作用。具体地说，我们将利用慢病毒系统建立IMP1过表达或敲除的Caco-2细胞系，从WT；Imp1fl/fl或VillinCre；Imp1fl/fl小鼠产生肠道小肠样，并使用葡聚糖硫酸钠(DSS)诱导WT；Imp1fl/fl或VC；Imp1fl/fl小鼠的急性肠道损伤。将使用Cyto-ID、培养细胞的跨上皮阻力测量和体内通透性分析来询问所有系统对自噬的影响。在目标2中，我们将定义 IMP1与自噬在肿瘤发生发展中的作用这将使用WT；Imp1fl/fl或VC；Imp1fl/fl小鼠中炎症相关癌症的AOM/DSS模型来实现，并将我们的IMP1报告模型(Imp1EGFP；DTR；Creer)与Apcflf1小鼠杂交以检查肿瘤起始，以评估IMP1阳性细胞在肿瘤起始中的作用。总之，这些研究将为IMP1在屏障功能和肿瘤发生/发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.