Epithelial mesenchymal transition in gastrointestinal homeostasis and disease

描述(申请人提供)：上皮-间充质转化(EMT)是一种高度保守的细胞程序，允许极化的、静止的上皮细胞转化为可运动的间充质细胞。EMT是发育过程中组织重塑事件的基础，但在成年人的伤口愈合、组织再生、器官纤维化、肿瘤侵袭和转移过程中，这一过程被重新参与。虽然EMT的生理和临床意义是压倒性的，但EMT的确切分子和功能特征仍然缺乏特征。绒毛蛋白是一种上皮细胞特异性肌动蛋白结合蛋白，在胃肠道、泌尿生殖系统和呼吸道大量表达。绒毛蛋白是一种多功能蛋白，它调节上皮细胞的可塑性和EMT等功能。用Villin基因敲除的小鼠所做的研究清楚地表明，Villin的缺失削弱了上皮细胞对调节EMT的信号的反应能力，导致伤口修复和细胞迁移不足。尽管有这些发现，维林如何驱动EMT的变化仍有待确定。在我们的初步研究中，我们首次证明了Villin定位于细胞核，并直接与转录辅助调节因子ZBRK1相互作用。此外，我们还报道了核绒毛蛋白激活转录因子Slug的表达，从而调节上皮的可塑性和EMT。最重要的是，我们的研究表明，绒毛蛋白离开刷状缘膜的错误定位是结直肠癌患者死亡率增加的预后因素。在本应用中设计的研究将检验我们的新假设，即绒毛蛋白的细胞质-核运输调节转录共调控因子ZBRK1的稳定性和/或周转，从而调节关键转录因子slug的表达以诱导EMT。其他研究旨在将绒毛蛋白的核异常定位与结直肠癌患者的转移联系起来。具体地说，本研究的目的是：(1)验证调节胞质-核转运和核保留的分子机制(S)；(2)鉴定绒毛蛋白-ZBRK1复合体及其在子宫内膜癌关键转录激活因子slug表达中的作用；(3)阐明核绒毛蛋白在调控子宫内膜转移和转移中的功能。我们提出的实验方法结合了机械性和功能性的生化、细胞和分子生物学研究，以及对Villin-/-、Villin+/+、ApcMin/Villin-/-、ApcMin/Villin+/+和严重联合免疫缺陷小鼠(SCID)小鼠的体内研究，使我们能够揭开EMT如何调控的复杂问题。建议使用两个独特的人类结肠癌组织微阵列资源进行其他研究，即来自参加III期MAX试验的患者的334个临床试验样本的队列和29个匹配的原发和转移性结肠癌样本的组织微阵列(TMA)。我们研究的长期目标是将我们的发现转化为临床结果，以诊断、预防和/或治疗纤维化和转移。

DESCRIPTION (provided by applicant): Epithelial-mesenchymal transition (EMT) is a highly conserved cellular program that allows polarized, immotile epithelial cells to convert to motile mesenchymal cells. EMT is fundamental for tissue remodeling events during development, but this process is re-engaged in adults during wound healing, tissue regeneration, organ fibrosis, tumor invasion and metastasis. While the physiological and clinical significance of EMT is overwhelming, the precise molecular and functional features of EMT remain poorly characterized. Villin is an epithelial cell specific actin-binding protein that is expressed in mos significant amounts in the gastrointestinal, urogenital and respiratory tracts. Villin is a multifunctional protein that regulates epithelial cell plasticity and EMT amongst other functions. Studies done with the villin knockout mice have clearly demonstrated that the absence of villin impairs the ability of epithelial cells to respond to signals that regulate EMT, resulting in a deficiency in wound repair and cell migration. Despite these findings, how villin actuates changes in EMT remains to be determined. In our preliminary studies, we demonstrate for the first time that villin localizes to the nucleus and interacts directly with a transcriptional co-regulator, ZBRK1. Furthermore, we report that nuclear villin activates the expression of the transcriptional factor Slug to modulate epithelial plasticity and EMT. Most importantly, our studies suggest that mislocalization of villin away from the brush border membrane is prognostic of increased mortality in patients with colorectal cancer. Studies designed in this application wil test our novel hypothesis that cytoplasmic-nuclear trafficking of villin regulates the stability and/or turnover of the transcriptional co-regulator ZBRK1, thereby modulating the expression of the key transcriptional factor Slug to induce EMT. Additional studies are designed to link the abnormal nuclear localization of villin with metastasis in patients with colorectal cancer. Specifically, the goal of the proposed studies are: (i) to validate the molecular mechanism(s) that regulates the cytoplasmic-nuclear trafficking and nuclear retention of villin; (ii) to characterize the villin-ZBRK1 complex and its role in the expression of Slug, a key transcriptional activator of EMT and; (iii) to elucidate the function of nuclear villin in the regulation of EMT and metastasis. The experimental approach we have proposed combines mechanistic and functional biochemical, cell and molecular biological studies together with in vivo studies in the villin-/-, villin+/+, ApcMin/villin-/-; ApcMin/villin+/+, and severe combined immunodeficiency mice (SCID) mice to allow us to unravel the complex question of how EMT is regulated. Additional studies are proposed using two unique human colon cancer tissue microarray resources namely, a cohort of 334 clinical trial specimens from patients enrolled in phase III MAX trial and a tissue microarray (TMA) of 29 matched primary and metastatic colon tumor specimens. The long-term goal of our studies is to translate our findings into clinical outcomes to diagnose, prevent and/or treat fibrosis and metastasis.